Chamber president Thomas Donohue said he was in Cuba to assess the economic changes taking place under President Raul Castro.
The US imposed an embargo on the communist-run island more than 50 years ago following the triumph of the Cuban Revolution.
Members of the Cuban community in the US have criticized the visit.
They accuse Raul Castro’s government of arresting political opponents and violating basic human rights.
Cuban-American Senator Robert Menendez said political opponents continued to be arrested “without justification,” in Cuba.
“Such conditions hardly seem an attractive opportunity for any responsible business leader,” Robert Menendez told the AP news agency.
Members of the trade delegation told AP that they were in Cuba to assess the trade possibilities in a post-embargo scenario.
However, the vast and politically-active Cuban community in the US has been strongly opposed to any change to trade ties with the communist-run island.
Since Fidel Castro handed power to his brother Raul in 2006, Cuba’s communist government has introduced a number of economic reforms.
Cubans are now allowed to own small businesses and to buy and sell cars and properties.
“We are very pleased to be here. We are learning a lot about the changes taking place in Cuba,” said Thomas Donohue.
He said that Cuba is now “fundamentally different in terms of the number of people that are operating under the private system”.
In December, President Raul Castro called for “civilized relations” with the US, saying the two countries should respect their differences.
The US should drop its demand for regime change and allow both sides to continue work on improving relations, Raul Castro said.
The US Chamber of Commerce delegation was welcomed to Havana on Tuesday afternoon by Foreign Minister Bruno Rodriguez and Trade Minister Rodrigo Malmierca Diaz.
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