Bacteria are able to interact with a number of macromolecules which act as opsonins or tissue-adherence factors. Because soluble fibronectin may be important factors. Because soluble fibronectin may be important in the phagocytic removal of bacteria and insoluble fibronectin may serve as a bridge between bacteria and host tissues, we have characterized the binding of soluble plasma fibronectin to Staphylococcus aureus as a first step to understanding these interactions. The binding of 125I-fibronectin to clinical and laboratory strains of S. aureus was studied. Bound fibronectin was separated from free fibronectin by centrifugation. Specific binding was determined by subtracting the amount bound in the presence of excess fibronectin from the total amount bound. We found that (i) fibronectin bound saturably, irreversibly, and noncovalently to S. aureus when the binding reaction was carried out at pH 7.4 or greater; (ii) S. aureus harvested in logarithmic phase of growth from media buffered to pH 8.4, and from brain-heart infusion media which demonstrated the greatest number of fibronectin-binding sites; (iii) high molecular weight dextrans, fibrinogen, cyanogen bromide fragment 7 of collagen, cationic proteins, dibromide fragment 7 of collagen, cationic proteins, dithiothreitol, and protein A did not alt er fibronectin binding to S. aureus; (iv) nonsaturable binding occurred below pH 7.0 with peak binding occurring at pH 5.8; and (v) there were marked differences in the amounts of fibronectin that bind to different strains of S. aureus. S. aureus ATCC 25923, when harvested in logarithmic phase of growth from tryptic soy broth and tested for fibronectin binding in (2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline, pH 7.4, had 7500 binding sites/organism with an apparent association constant of 5.6 X 10(9) M-1.