Staphylococcus aureus infection triggers polyclonal B-cell activation. It was sought to further characterize the hypergammaglobulinemia seen in Staphylococcus aureus infection, focusing on the significance of protease-specific B-cell responses. Sera from mice infected with Staphylococcus aureus wild-type strain 8325-4 and two of its isogenic mutants devoid of protease expression were analyzed for the occurrence of polyclonal B-cell activation and the presence of specific antibodies against a set of exoproteases and superantigens. Furthermore, the functional properties of anti-V8-protease antibodies were analyzed in vitro. Polyclonal activation was manifested by increased levels of total serum IgG and IgM in all infected animals and by antibodies to staphylococcal toxins and aureolysin whether these antigens were present in the inoculate or not. Importantly, Staphylococcus aureus mutant lacking the V8-protease did not trigger a response against this enzyme. In contrast, strains expressing the V8-protease elicited V8-protease antibodies, proving the antigen-specific nature of this response. In vitro tests revealed that these antibodies had the capacity to inhibit the V8 protease activity in a dose-dependent manner. It was concluded that exposure to Staphylococcus aureus, in addition to a massive polyclonal B-cell response, gives rise to production of exoprotease-specific antibodies displaying functional properties.