S. aureus strain ISP 546 was selected (of 55 strains tested) to define optimal conditions for expression of vitronectin binding. High binding was expressed when the strain was grown on blood agar and in Todd-Hewitt broth. Binding was optimal in the 6.0 to 7.2 pH range and was unaffected by divalent cations and ionic strength. Binding was partially inhibited by D-mannose, heparin, types I and IV collagen, fibronectin, fibrinogen and vitronectin, but was not affected by other carbohydrates or glycoproteins tested. Cell surface binding components were extracted with the aid of 1 M LiCl (pH 5.0) from strain ISP 546 grown in Todd Hewitt broth. Vitronectin binding proteins were purified by affinity chromatography on heparin-Sepharose. Fractions inhibiting binding of 125I-labelled vitronectin to strain ISP 546 were eluted by 0.01 M NaOH, dialysed, concentrated and subjected to SDS-PAGE. Silver staining revealed one major band (70 kDa) and two minor bands (34 and 36 kDa).