In an earlier report, we had described the isolation and characterization of autolysis-defective mutants of Staphylococcus aureus (N. Mani, P. Tobin, and R.K. Jayaswal, J. Bacteriol. 175:1493-1499, 1993). In the study reported here, an autolysis-defective mutant showed attenuated virulence in a rat model of experimental endocarditis, supporting the role of autolysins in pathogenicity. Transmission electron micrographs of the mutant cells revealed a rough outermost surface as compared with the parent strain, ISP2018. Treatment of mutant cells with lysozyme, proteases, and lipase failed to alter this rough appearance. Physical and genetic data locate the site of mutation between the omega 1100 and ilv loci on the S. aureus chromosome.