Staphylococcal alpha-toxin targets several cell types which are important components of cardiac vegetations in endocarditis, including platelets, erythrocytes, and endothelial cells. We evaluated the in vivo role of Staphylococcus aureus alpha-toxin in experimental endocarditis by using isogenic strains differing in the capacity to produce functional alpha-toxin, including 8325-4 (wild-type strain), DU-1090 (a mutant strain with allelic replacement of the alpha-toxin gene [hla]), DU1090(pH35L) (a mutant strain producing a target cell-binding but nonlytic toxin), DU1090(pDU1212) (a variant of DU1090 carrying the cloned hla gene on a multicopy plasmid), and DU1090(pCL84::hla) (a variant of DU1090 with a single copy of the hla gene cloned into the chromosomal lipase locus). In vitro, wild-type alpha-toxin (from parental strain 8325-4) extensively lysed both erythrocytes and platelets. In contrast, mutant alpha-toxin [from strain DU1090(pH35L)] lysed neither cell type. Following exposure to the wild-type alpha-toxin, platelet lysates were found to contain microbicidal activity against Bacillus subtilis (but not against Micrococcus luteus), as well as against the parental and alpha-toxin variant S. aureus strains noted above. Furthermore, lysate microbicidal activity was heat stable, neutralized by polyanionic filters or compounds, and recoverable from anionic filter membranes by hypertonic saline elution. These characteristics are consistent with those of cationic platelet microbicidal proteins (PMPs). Reverse-phase high-pressure liquid chromatography and polyacrylamide gel electrophoresis confirmed the presence of three distinct PMPs (1, 2, and 3) in platelet lysates. In experimental endocarditis, the two variant staphylococcal strains producing either minimal alpha-toxin or nonlytic alpha-toxin in vitro [strains DU1090 and DU1090(pH35L), respectively] exhibited significantly lower virulence in vivo than the parental strain (decreased intravegetation staphylococcal densities). Paradoxically, the two variant staphylococcal strains producing alpha-toxin at supraparental levels in vitro [strains DU1090(p1212) and DU1090(pCL84::hla)] also exhibited significantly decreased induction rates and intravegetation staphylococcal densities in experimental endocarditis versus the parental strain. The reduced in vivo virulence of the latter variant staphylococcal strains could not be explained by differences in bacteremic clearance or initial adherence to sterile vegetations (compared to the parental strain). These findings suggest that the reduced virulence exhibited by the variant staphylococcal strains in this model was related to pathogenetic events subsequent to bacterial adherence to the damaged endocardium. Excess intravegetation secretion of alpha-toxin, leading to increased PMP release (secondary to either increased platelet secretion or lysis), may well explain the reduced virulence observed in experimental endocarditis.