Bone sialoprotein is selectively bound by Staphylococcus aureus cells isolated from patients suffering from infections of bone tissue [Rydén, C., Maxe, I., Franzén, A., Ljungh, A., Heinegård, D. & Rubin, K. (1987) Lancet II, 514]. In the present communication the binding of bone sialoprotein to staphylococcal cells is characterized in more detail. 125I-Labelled bone sialoprotein bound to suspended staphylococcal cells in a time-dependent, saturable and reversible manner. Binding was inhibited by unlabelled bone sialoprotein and by an amino-terminal CNBr fragment of bone sialoprotein that did not contain the eukaryotic cell-binding site. Binding was furthermore inhibited by lysates obtained from Escherichia coli lysogens carrying a lambda gt11 phage-encoding bone sialoprotein. In contrast, binding was not inhibited by a bacterial lysate from an osteopontin lambda gt11 lysogen, nor by N-linked oligosaccharide isolated from bone sialoprotein or by proteoglycan from rat chondrosarcoma containing clustered O-linked oligosaccharides of the same structure as those of bone sialoprotein. These results indicate that the major staphylococcal-binding site resides in the bone sialoprotein core protein and not in the carbohydrate side chains. No inhibition of bone sialoprotein binding could be detected for whole human serum or purified plasma proteins such as fibronectin, fibrinogen and IgG. Likewise, staphylococcal protein A or rat collagen type I did not inhibit the binding of bone sialoprotein. The latter results indicate that the binding site for bone sialoprotein on staphylococcal cells was not any of the hitherto described staphylococcal cell-surface proteins. Binding data indicated an average of 1000 bone-sialoprotein-binding sites/bacterial cell.