To improve alkaline protease producing strain by protoplast fusion, a strain of Bacillus sp. was treated with 100㎍/㎖ NTG (N-methyl-N`-vitro-N-nitrosoguanidine) for 45 minutes and mutants of Bacillus subtilts Arg^-, Try^-, His^- and Ade^- were isolated. The frequency of protoplast formation was about 99%, when teas of exponential phase were treated with 200㎍/㎖ lysozyme at 42℃ for 10∼30 minutes. In a regeneration medium containing 0.3M sodium succinate, 2.0% polyvinylpyrrolidone, 0.5% casamino acid, 10mM MgCl₂ and 20mM CaCl₂, regeneration frequency cf the isolated Bacillus subtilis strains was 25.2%. The fusion frequency between mutant, was from 2.1×10^(-5) to 8.1×10^(-5) under optimum condition.