A flotilla of four boats, organized by the Democracy Movement, traveled from Key West to a point about 12 miles from Havana, where they tossed flowers and 41 white Styrofoam crosses into the water.
They used mirrors to flash messages toward El Malecon, a popular sea wall in Havana where Cubans gather.
"It's a symbolic way to unite a people divided by a wall of intolerance," said Ramon Saul Sanchez, a Democracy Movement leader. "It is a means to tell the Cuban people that we don't forget them."
Three planes flown by members of the exile group Brothers to the Rescue passed over the flotilla before dropping six smoke markers above the spot where four compatriots were shot out of the skies by Cuban jet fighters in February 1966.
The plumes of smoke extended 3,000 feet (900 meters)into the air, said Jose Basulto of Brothers to the Rescue.
"It's always a moment of sadness every time I go out there," Basulto said. "Every time the occasion calls for it, we will go there to remember our fallen, especially the ones who have been murdered by the government of Cuba."
Coast Guard monitors ceremony
The 41 Cubans who died were aboard the tugboat 13 de Marzo, which sank July 14, 1994, as it was being chased by Cuban vessels. Those on board were trying to flee to Florida.
On Friday night, it appeared as if the U.S. Coast Guard might try to stop the flotilla from leaving port when Democracy Movement leaders refused to give assurances that the boats would not try to enter Cuban waters.
"We always reserve our right under the law ... to enter our homeland, our territory, our waters," a defiant Sanchez said.
Under a U.S. policy begun in 1996 after the Brothers to the Rescue planes were shot down, the Coast Guard can seize any vessel that heads from Florida toward Cuban territory without a permit.
But in the end, the boats were allowed to leave, escorted by a Coast Guard cutter. The number of boats in the flotilla was scaled back from 10 to four, and they were required to stay together.
The boats did not enter Cuban waters, and no Cuban patrol boats were seen. Along Havana's waterfront, no special security measures were visible.
Reuters contributed to this report.