Abstract: The factors that influenced the formation of protoplasts of two nematode-trapping fungi,including culture media,mycelial stages,lysing enzymes and its dilution buffers,were studied and the optimized technique for protoplast preparation was determined.By the optimized protocol,the protoplast yields of the two fungi,Arthrobotrys vermicola and Monacrosporium sphaeroides were 0.5×107—1.0×107mL-1,and 0.8×107—1.5×107mL-1 respectively.The regeneration of protoplasts was affected not only by the generation method,but also by the nutrients,the osmetic stablizer and its concentration in the regeneration media.Additionally,the process of protoplast generation and regeneration was observed and described in detail.