Protoplasts were isolated enzymatically from the calli derived from leaf primordia of adult Japanese persimmon (Diospyros kaki L. cv. Jiro). They were cultured on revised KM8p medium with modified concentrations of growth regulators, ammonium nitrate, and glucose. The optimum concentrations of NAA, zeatin, ammonium nitrate and glucose appeared to be 10 muM, 1 muM, 150 mg/l and 0.5 M, respectively. With the best combination of these components, the initial plating density of protoplasts was reduced to one fifth (1 x 10(5) protoplasts/ml) that required in a previous paper (TAO et al. 1991). The microcalli thus obtained showed most vigorous growth on a modified KM8 medium with the concentration of ammonium nitrate reduced to 150 mg/l supplemented with 1 muM zeatin plus either 1 muM IAA or 1 muM NAA. On this medium, most microcalli grew to more than 5mm in diameter in 6 weeks. It was demonstrated that plantlets could be obtained from the protoplast-derived calli. There was no difference in the isozyme banding patterns of three enzymes-glucose phosphate isomerase (GPI), phosphoglucomutase (PGM), and malate dehydrogenase (MDH)-between the plantlets from protoplasts and shoot tips. However, differences were observed in the color of young leaves between these two plantlet types.