Lycopersicon pennellii shoots, cultured in vitro for more than a year (type I plants) produced few viable protoplasts in contrast to shoots cultured in vitro for less than five months (type II plants). Ethylene production of both plant types was compared. The low viability of plant type I protoplasts could be correlated with high ethylene production and an increased cell sap osmolality. The ethylene action inhibitor silver thiosulphate improved protoplast yield and viability, especially when using donor tissue, germinated and cultured on medium containing silver thiosulphate (type III plants). Moreover, the choice of cell wall degrading enzymes influenced protoplast viability, since ethylene release was significantly lower using Cellulase R 10 than Cellulysin. All improvements together resulted in an efficient protocol for the isolation and regeneration of Lycopersicon pennellii protoplasts.