To investigate the fusion of bacterial protoplasts, two auxotrophic mutants of Bacillus subtilis were isolated after treatment with nitrosoguanidine. Two auxotrophs were uracil-auxotroph and tryptophan-auxotroph. Back mutation frequencies of ura- and trp- -auxotrophs were 2.4×10^(-8) and less than 1.0×10^(-8) respectively. The optimal pH and temperatnre of protoplast formation with lysozyme were 6.5 and 30℃. The optimal lysozyme concentration was 200㎍/㎖. The regeneration frequency of protoplast was 3.3%.