A method for the formation and regeneration of protoplasts of several strains of the chestnut blight fungus, Cryphonectria parasitica, is presented. The procedure utilizes cellophane membranes for growth and employs centrifugation for separation of protoplasts from hyphal fragments. Yields averaged 8.04 x 10(6) protoplasts per membrane. Regeneration frequencies were 40-50% with a soft-agar overlay. These protoplasts are suitable for use in experiments designed to determine the role of dsRNA in hypovirulence of C. parasitica.