In order to obtain a highly urea-resistant Staphylococcus aureus V8 protease for producing pep tide hormones by recombinant DNA technology, we constructed double and triple mutants of recombinant S. aureus V8 protease by combining previously isolated urea-resistant mutations D44E, N71S and K147R. The double mutants were more stable than the single mutants, and the triple mutant was even more stable than the double mutants, showing that the effect caused by the three mutations was additive. The half life of the triple mutant (V8 Delta 53-U158) in 5 M urea was 21 times longer than that of S. aureus V8 protease. This increased stability by combining each urea-resistant mutation was only effective to urea, and not to 0.1% sodium dodecyl sulfate nor to heat at 50 degrees C. The triple mutant was applied to the digestion reaction of a fusion protein containing the human calcitonin precursor peptide (hCT[G]). It was demonstrated that the fusion protein was efficiently digested in the presence of over 5 M of urea, and that the triple mutant could release 2 times more hCT[G] than could S. aureus V8 protease.