Isogenic strains of Staphylococcus aureus, differing in fibronectin binding, were constructed for studies of the contribution of fibronectin binding to the in vivo pathogenesis of staphylococcal disease. Mutagenesis of S. aureus 879R4S was accomplished by mating with Enterococcus faecalis FA378 that carried the transposon Tn918. Four low-fibronectin-binding mutants were identified that bound 24- to 35-fold less fibronectin than the parent strain did. A spectinomycin-resistant strain, R4SSp, was transduced by a bacteriophage (80 alpha) lysate propagated on a low-binding mutant of 879R4S to produce R4SSp/1536, which bound less fibronectin, contained a single copy of the transposon, and grew on spectinomycin-containing medium. Using a rat model of endocarditis, we determined the distribution of S. aureus R4SSp and its transductant in normal and cardiac catheterized rats. Cultures of heart tissue showed that catheterized rats challenged with the fibronectin-binding parent strain had over 250-fold more organisms in the left heart than did rats challenged with the low-binding transductant. The ability to bind fibronectin had no effect on the number of S. aureus cells cultured from other tissues. These data suggest that the ability of S. aureus to bind fibronectin is an important factor in establishing adherence to damaged heart valves in vivo.