Gram-positive pathogenic bacteria display proteins on their surface that play important roles during infection. In Staphylococcus aureus, these surface proteins are anchored to the cell wall by two sortase enzymes, SrtA and SrtB, that recognize specific surface protein sorting signals. The role of sortase enzymes in bacterial virulence was examined using a murine septic arthritis model. Intravenous inoculation with any of the Delta(srtA), Delta(srtB) or Delta(srtAB) mutants resulted in significantly increased survival and significantly lower weight loss compared with the parental strain. Mice inoculated with the Delta(srtA) mutant did not express severe arthritis, while arthritis in mice inoculated with the Delta(srtB) mutant was not different from that seen in mice that were infected with the wild-type parent strain. Furthermore, persistence of staphylococci in kidneys and joints following intravenous inoculation of mice was more pronounced for wild-type and Delta(srtB) mutant strains than for Delta(srtA) or Delta(srtAB) variants. Together these results indicate that sortase B (srtB) plays a contributing role during the pathogenesis of staphylococcal infections, whereas sortase A (srtA) is an essential virulence factor for the establishment of septic arthritis.