An attempt was made to produce protoplasts with a high potential of cell colony formation from sugarcane cell suspensions through an enzyme treatment. Those suspensions were intiated from fresh leaf calli as well as leaf and young panicle calli each on agar media. The key procedures presented in this study are summarized as follows: (1) To use calli with a high potentiality of plant regeneration in rapidly growing phase, i.e. 10-20 days after transfer; (2) To treat with enzyme the cell suspensions subcultured only once after the last subculture on agar media, or the calli o agar media directly; (3) To incubate the cell-enzyme mixture under shaking at 30-60 rpm at 32℃ for 4-10 hr; (4) For effective formation of cell colonies, an AA liquid medium is most suitable, where reduction of osmotic pressure is not usually necessary; and (5) For morphogenic calli formation, formed calli must be transferred to another medium, such as 1/2 MS, MS, B5 or N6 medium supplemented with 5 × 10^-6M 2,4-D and 10^-6M NAA. In about a month after the treatment, a large number of cell colonies consisting of small, tightly packed cells were produced. They eventually developed into morphogenic calli differentiating roots.