Adm Stavridis said the fleet would support counter-terrorism efforts
The United States Fourth Naval Fleet has resumed operations off the Caribbean and the length of Latin America for the first time since 1950.
The US Department of Defense says the measure is aimed at building confidence and trust in the region by focussing on common threats.
But some leaders in the region are concerned by the move.
Bolivian President Evo Morales has dubbed the initiative the Fourth Fleet of intervention.
Cuban former President Fidel Castro has written that the aircraft carriers were a threat that would sow terror.
Based in Florida, the fleet will include various ships, including aircraft carriers and submarines and will patrol waters from the Caribbean and along the length of Latin America's coastline.
The fleet will help fight drug traffickers and work on humanitarian and diplomatic exercises.
The head of the US Southern Command, Adm James Stavridis, told Congress in March that he supported plans for a fleet to back counter-terrorism operations.
Chief of Naval Operations Adm Gary Roughead said: "Re-establishing the Fourth Fleet recognises the immense importance of maritime security in the southern part of the Western Hemisphere," said
But the growing number of anti-US leaders in Latin America are asking questions about the new measure, says BBC Latin America correspondent Daniel Schweimler.
Washington has become concerned by increasing anti-US rhetoric and the build-up of military forces, especially in Venezuela, our correspondent adds.
The US Navy also has operational fleets in the Pacific, the Gulf and off the coast of Asia.
The Fourth Fleet was created during the Second World War to combat German U-boats targeting merchant shipping lines in the South Atlantic.
But when the war ended the fleet was deemed to be no longer necessary and was disbanded in 1950.
Correction 23 July 2008: An earlier version of this story quoted Adm Stavridis as saying that he supported plans for a fleet led by a nuclear aircraft carrier to back counter-terrorism operations. This was incorrectly reported as he did not say this in his testimony to Congress.