In this study the specific binding of soluble type I collagen to several strains of Staphylococcus aureus was investigated. Both type I procollagen and soluble, pepsin-treated, lathyritic rat skin type I collagen bound to these bacteria in a manner which could be blocked by the addition of gelatin to the binding assay. Saturation binding studies showed more than one class of binding sites for [125 I]-lathyritic rat skin collagen to be present with each bacterium of the Cowan I strain containing approximately 135 high affinity sites with an apparent KA of 2.3 X 10(7)M-1. Like Cowan I strain, American Type Culture Collection (ATCC) strain 25923 also bound type I collagen. IgG inhibited collagen binding in a dose dependent manner. This observation together with the finding that the protein A-deficient Wood strain did not bind collagen suggested that protein A might be the collagen binding site. However, failure of protein A-Sepharose to bind soluble collagen or protein A in solution to inhibit binding of collagen to Cowan I cells suggests that bacterial protein A does not mediate the binding. Addition of fibronectin to the binding assay did not affect the level of collagen binding, suggesting a) that the collagen binding site is different from the fibronectin binding site and b) that fibronectin does not mediate the binding of collagen to these cells. These results demonstrate a new example of bacterial binding to an extracellular matrix protein and suggest a possible mechanism whereby Staphylococcus aureus may adhere to mammalian tissue.