It was a good night for Argentine clubs as 1985 champions Argentinos Juniors beat America of Mexico 3-1 in Group Three at the Diego Armando Maradona ground with two goals by Paraguayan striker Santiago Salcedo.
Independiente scored second-half goals by striker Facundo Parra, midfielder Cristian Pellerano and substitute Andres Silvera for their eighth win over Penarol in 14 meetings since South America's Champions League equivalent was first played in 1960.
"He asked us to play a little further upfield and keep more possession," Independiente captain Eduardo Tuzzio, voted man of the match, told reporters summarising coach Antonio Mohamed's halftime team talk.
"I think this was the best the team have done since I've been at the helm," he added.
Independiente's improvement paid immediate dividends when Parra put them ahead less than two minutes into the second half with a volley from midfielder Patricio Rodriguez's chipped pass.
Goalkeeper Hilario Navarro had to save sharply from Juan Manuel Olivera on the left to prevent an equaliser and Pellerano scored Independiente's second with a low shot from 25 metres following a clearance.
Penarol, five-times winners but a long way from the team voted by FIFA's statistics body as South America's best of the 20th century, cleared two more chances off the line before Silvera completed the win from a neat layoff by fellow substitute Leonel Nunez.
Ecuador's LDU Quito, winners in 2008, and Argentine debutants Godoy Cruz are the other teams in Group Eight.
