A rapid method for isolation and culture of coffee leaf protoplasts was developed. For the isolation, less than one month old leaves from two lines of Coffea arabica, five lines of C. canephora, a sexual hybrid line of C. arabica × C. canephora from Cenicafé (Colombia) and another sexual hybrid (Arabusta) from Gerdat (France) were treated with several enzyme combinations. Four hour treatment with Cellulase R-10 or Cellulase A 2230 (3%) in combination with Pectolyase Y-23 (0.5%) in 0.6M Mannitol and pH 5.8, gave the best yields in all lines and species tested.