A method was developed for protoplast isolation and culture from subcultures of apple (Malus pumila MILL. var. domestica SCHNEID.) calluses induced originally by anther culture. Viable protoplasts were obtained by using an enzyme solution containing 2% cellulase "Onozuka" R-10, 0.1% pectolyase Y-23 and 0.7M mannitol (pH5.8) and the optimum incubation time was 2.5hr. Protoplasts thus obtained were cultured in a modified 8p medium containing benzyladenine (BA) instead of zeatin without the use of coconut water. Within 1 week of culture, the protoplasts initiated cell division and the addition of fresh medium induced a marked increase in the number of cell colonies. The optimum plating density was 1-2xl05 protoplasts/ml and higher densities of protoplasts tended to inhibit colony formation. The induced colonies were transferred onto media solidified with agar. The colonies then formed calluses during 1 month, but no shoot or root was formed.