To investigate optimal conditions for the protoplast formation and regeneration of Phanerochaete chrysosporium, preparations of three enzymes were used to liberate protoplasts from its 20 hrs-old mycelium on cellophan membrane covered agar media. Novozym 234 alone with 0.6M sucrose was the most effective for isolation of protoplasts from the mycelium with 3hrs incubation time at $39^{circ}C$ in shaking condition of 120 rpm. The poly-R medium stabilized with 0.6M mannitol was the best for regeneration of the protoplasts.