Longitudinal sections containing cortical cells taken from stem internodes of a hybrid betweenLycopersicon esculentum andSolanum lycopersicoides were used as tissue sources for enzymatic protoplast isolation. Greenhouse and growth room-grown plants 4-8 weeks after rooting could be used as sources of donor tissue. Protoplasts from these tissues divided within 2-4 days of culture and numerous microcalli formed within 30 days. The shoot regeneration frequency of protoplast-derived calli was in the order of 60%. More than 100 regenerated plants which appear phenotypically normal have been established in soil.