Adhesion of Staphylococcus aureus to human endothelial cells is implicated in the pathogenesis of invasive staphylococcal disease. The adhesion to endothelial cells of isogenic mutants defective in defined surface structures was studied. Three strains of S. aureus defective in fibronectin-binding proteins FnBPA and FnBPB showed reduced adhesion. This was fully restored by complementation of a FnBPA- FnBPB- mutant derived from strain 8325-4 with a multicopy plasmid encoding FnBPA or FnBPB. Adhesion of mutants defective in other surface structures was unaffected. Anti-fibronectin antibodies blocked adhesion of 8325-4 to endothelial cells, while adhesion of strains 8325-4, P1 and five clinical isolates was inhibited by the recombinant form of the binding domain of FnBPB (rFNBD) from Streptococcus dysgalactiae. Adherence of bacterial aggregates resulting from the presence of purified fibrinogen was also inhibited by rFNBD protein. Three strains of S. aureus defective in FnBPA and FnBPB were not internalized by endothelial cells. S. aureus FnBPs mediate adhesion to human endothelial cells and are required for subsequent internalization, interactions of potential relevance to pathogenesis and treatment.