The degree of autolysis and lytic enzyme production in the culture filtrate of Rhizopus oryzae was investigated. The formation of protoplast by using autolytic enzymes from Rh. oryzae was also attempted. Protoplasts were liberated from Rh. oryzae mycelium by lytic enzymes present in autolytic-phase culture filtrate. Maximum release of chitosanase and proteolytic enzyme into culture filtrate during autolysis was corresponded to maximum protoplast-liberating activity. High yields of protoplasts were obtained from 10 hr-age of Rh. oryzae mycelium with 0.5 M mannitol as osmotic stabilizer. The optimum temperature and pH for mycelium digestion were $25{sim}30^{circ}C$ and $6.0{sim}6.5$ respectively. The mycelium of the 18 hours cultures were treated with autolytic enzyme in same volume of osmotic stabilizer at $30^{circ}C$ for 5 hours and then it was confirmed by scanning electoron microscope that protoplast were produced beside the digesting cell wall of the fungi.