In 1801, he was appointed as an aide by President Thomas Jefferson, whom he knew personally through Virginia society in Albemarle County. Lewis resided in the White House, and frequently conversed with various prominent figures in politics, the arts and other circles. Originally, he was to provide information on the politics of the United States Army, which had seen an influx of Federalist officers as a result of John Adams's "midnight appointments." When Jefferson began to formulate and to plan for an expedition across the continent, he chose Lewis to lead the expedition.
Jefferson selected Captain Meriwether Lewis to lead the proposed expedition, afterwards known as the Corps of Discovery. Lewis became intimately involved in planning the expedition and was sent by Jefferson to Philadelphia, Pennsylvania, for instruction in cartography and other skills for making scientific observations. In June 1803, Jefferson provided Lewis with basic objectives for the mission, focusing on the exploration of the Missouri river and any related streams which might provide access to the Pacific Ocean. Lewis concluded the expedition would benefit from a co-commander and, with Jefferson's consent, offered the assignment to his friend and former commanding officer, William Clark. Clark and Lewis were both relatively young and adventurous and had shared experience as woodsmen-frontiersmen and Army officers. However the two men were quite different in education and temperament. Lewis was introverted and moody while Clark was extroverted, even-tempered and gregarious. Lewis, who had a better education, possessed a philosophical and speculative outlook and was at home with abstract ideas. Clark was more pragmatic and practical. Because of bureaucratic delays in the U.S. Army, Clark officially only held the rank of Second Lieutenant at the time, but Lewis concealed this from expedition members and shared the leadership of the expedition, always referring to Clark as "Captain". 
Lewis departed Pittsburgh for St. Louis—the capitaal of the new Louisiana Territory—via the Ohio River in the summer of 1803, gathering supplies, equipment, and personnel along the way. Between 1804 and 1806, the Corp of Discovery explored thousands of miles of the Missouri and Columbia River watersheds, searching for an all-water route to the Pacific Ocean. Generally sharing leadership responsibilities with William Clark, although technically the leader, Lewis led the expedition safely across the Rocky Mountains to the Pacific and back, with the loss of just one man, Charles Floyd, who died of apparent appendicitis. In the course of the journey, Lewis observed, collected, and described hundreds of plants and animal species previously unknown to science. The expedition was the first point of Euro-American contact for several Native American tribes; through translators and sign language, Lewis conducted rudimentary ethnographic studies of the peoples he encountered, even as he laid the groundwork for a trade economy to ensure American hegemony over its vast new interior territory.
On August 11, 1806, near the end of the expedition, Lewis was shot in the left thigh by Pierre Cruzatte, a near-blind man under his command, while both were hunting for elk. His wound hampered him for the rest of the journey.
Lewis was a Freemason, initiated, passed and raised in Door To Virtue Lodge No. 44 in Albemarle, VA between 1796 and 1797. On August 2, 1808, Lewis and several of his acquaintances submitted a petition to the Grand Lodge of Pennsylvania in which they requested a dispensation to establish a lodge in St. Louis. Lewis was nominated and recommended to serve as the first Master of the proposed Lodge, which was warranted as Lodge No. 111 on September 16, 1808.
In September 1809 Lewis was traveling to Washington D.C. to answer complaints about his actions as governor. While en route he stopped at a tavern called Grinder's Stand, about 70 miles (110 km) from Nashville, Tennessee, on the Natchez Trace on October 10th. The next morning Lewis was found with multiple gunshot wounds; he died shortly after sunrise.
While it is generally accepted by modern historians that his death was a suicide, there is some debate concerning this. Mrs Grinder the tavern-keeper's wife claimed that Lewis was acting strangely the night before his death. She claimed that during the dinner Lewis would stand and pace about the room while speaking to himself. His manner of speech struck her as if he was speaking as a lawyer. After Lewis had retired for the evening Mrs Grinder continued to hear Lewis talking to himself. At some point during the night Mr Grinder heard multiple gunshots and what she believed to be a voice asking for assistance. She claimed to be able to see Lewis through the slit in the door crawling back to his room. Why Mrs Grinder did not inquire as to the status of Lewis or the source of the gunshots has never been explained. The next morning when she sent for Lewis's servants they found him near death.
Later when Clark and Jefferson were informed of Lewis's death both accepted the belief it was suicide, his family however contended it was murder. In later years a court of inquiry was held charging the tavern-keeper with Lewis's death. The inquiry was dropped for lack of evidence or motive.
The explorer was buried not far from where he died. He is honored today by a memorial along the Natchez Trace Parkway.
For many years, Lewis's legacy was overlooked, inaccurately assessed, and even tarnished by his alleged suicide. Yet his contributions to science, the exploration of the Western U.S., and the lore of great world explorers, are considered incalculable.
Four years after Lewis's death, Thomas Jefferson wrote:
Of courage undaunted, possessing a firmness and perseverance of purpose which nothing but impossibilities could divert from its direction, ... honest, disinterested, liberal, of sound understanding and a fidelity to truth so scrupulous that whatever he should report would be as certain as if seen by ourselves, with all these qualifications as if selected and implanted by nature in one body for this express purpose, I could have no hesitation in confiding the enterprise to him.
Jefferson also stated that Lewis had a "luminous and discriminating intellect."
The alpine plant Lewisia (family Portulacaceae), popular in rock gardens, is named after Lewis, as is Lewis's Woodpecker. Geographic names that honor him include Lewis County, Idaho, Lewis County, Tennessee; Lewisburg, Tennessee; Lewiston, Idaho; Lewis County, Washington; the U.S. Army fort Fort Lewis, Washington, the home of the US Army 1st Corps (I Corps), and especially Lewis and Clark County, Montana, the home of the capital city, Helena.
The US Navy Polaris nuclear submarine USS Lewis and Clark was named for him and William Clark.