Three different urea-resistant Staphylococcus aureus Y8 protease derivatives (V8 Delta 53-U1, V8 Delta 53-U5 and V8 Delta 53-U8) were obtained by random mutagenesis using a polymerase chain reaction (PCR) method. From DNA sequence analysis of the mutants, three different amino acid substitutions, D44E, N71S and K147R, were identified. The half-lives of these mutant enzymes were 3 to 5 times longer than that of the wild type in the presence of 5 M urea, and they also showed increased stability to sodium dodecyl sulfate. Measurements of the kinetic parameter values of each mutant enzyme indicate that these mutations substantially conferred the stability to these denaturants without markedly changing their kinetic parameter values. In addition, we demonstrate that these mutant enzymes were applicable to digestion of a human calcitonin fusion protein produced in Escherichia coli.