Thrombin-induced platelet microbicidal protein (tPMP) is secreted by rabbit platelets following thrombin stimulation, and it kills common endovascular pathogens in vitro, including Staphylococcus aureus. Therefore, pathogens which exhibit tPMP resistance in vitro possess a potential survival advantage in vivo at sites of endovascular damage. We generated an isogenic S. aureus strain pair, differing in tPMP susceptibility, by transposon (Tn551) mutagenesis of a tPMP-susceptible (tPMPs) parental strain (ISP479) to derive a stably tPMP-resistant (tPMPr) strain, ISP479R. ISP479 and ISP479R were equivalent in vitro in the following phenotypes: biotyping, antiobiograms, platelet adherence and aggregation, growth kinetics, cell wall-associated protein A expression, and fibrinogen binding. Genotypic comparisons of chromosomal DNA of strains ISP479 and ISP479R following restriction endonuclease digestion revealed indistinguishable pulsed-field gel electrophoretic patterns. The genotype exhibited by strain ISP479R was linked to the tPMP-resistant phenotype, as it was transducible into the initially tPMP-susceptible parental strain, ISP479. Southern hybridization verified the presence of a single copy of Tn551 in the same chromosomal restriction site of both ISP479R and tPMPr transductants of ISP479. The correlation of in vitro tPMP susceptibility phenotypes with the ability to induce experimental endocarditis (a prototypical endovascular infection) was evaluated. Despite equivalent rates of endocarditis induction, animals infected with strain ISP479R achieved significantly higher vegetation bacterial densities over a 7-day post-challenge period than did animals infected with strain ISP479. These data suggest that tPMPr microbial strains have a selective advantage in experimental staphylococcal endocarditis. Furthermore, the major impact of tPMP resistance upon endocarditis pathogenesis appears to involve a postvalvular adherence event(s), most probably by facilitating bacterial proliferation within vegetations.