Protoplasts were isolated from fruit-bodies of Agaricus bisporus, and highest yields were derived from basidia. When gill fragments were treated with a combination of Novozyme 234, chitinase, and cellulase Onozuka R-10, and with 0.35 M KCl as the osmotic stabilizer, high yields (3-4 x 10(7) protoplasts/g fresh wt gills) were obtained within 1 h of incubation. About 20-30% of protoplasts regenerated in a solid MMNC medium. Investigation of specific activities of glucose-6-phosphate dehydrogenase and mannitol dehydrogenase indicated a highly active pentose phosphate pathway and a good capacity for mannitol synthesis in protoplasts, as well as in other cells of fruit-bodies of the species. The simple and efficient procedure provides a new approach for further investigation of the mushroom, and possibly of other basidiomycetes with hemiangiocarpus, by use of protoplasts.