Protoplasts were produced from conidia of Collectotrichum gloespoiodes f. sp. aeschynomenc, a fungal plant pathogen of Aeschynomene virginica, during treatment with Novozym 234 or a mixture of chitinase and beta-glucuronidase after pretreatment with 2-mercaptoethanol. Protoplasts were optimally stabilized in 1.2 M mannitol after release from conidia but regenerated and reverted to hyphae optimally on 0.7 M sucrose. Approximately 84% of the protoplasts regenerated cell walls and reverted to hyphal colonies on 0.7 M sucrose. The osmotic stabilizer and molarity of the stabilizer affected regeneration and reversion to colonies. Microscopic studies of the nuclear content of conidia protoplasts showed that the number of nuclei in protoplasts was similar to the number of nuclei in conidia from which they were produced. Of the 209 colonies grown from reverted protoplasts, all were as pathogenic to A. virginica as the wild-type parent, and all resembled the wild-type strain from which they were produced. The development of an efficient technique to produce protoplasts enables future research on the genetics of the fungus. (Copyright (c) 1990, by The New York Botanical Garden, Bronx, NY.)