Temperature-sensitive (ts) mutants of Staphylococcus aureus were isolated after mutagenesis with nitrosoguanidine and two cycles of enrichment with Penicillin G and D-Cycloserine. The mutants expressed tight, coasting, and leaky phenotypes on solid media. In broth, however, most exhibited coasting for a limited number of generations. The reversion frequency of selected ts mutants was less than 10(-6). Intraperitoneal (i.p.) immunization with ts mutant G/1/2 conferred significant protection (0 dead/6 total vs. 7/7, immunized vs. control; p = 0.0006) from lethal i.p. challenge with the parental wild-type (wt) S. aureus suspended in 5% porcine mucin, performed 28 days after i.p. administration of 10(8) colony-forming units. Protection induced by mutants of coasting phenotype was higher and lasted longer than that induced by mutants of the tight phenotype. The results of this study demonstrate that ts mutants of S. aureus can be obtained and that ts mutants are able to induce protective immunity from subsequent challenge with the parental wt strain.