Staphylococcus aureus binds to purified fibronectin in solution and may bind to fibronectin present in wound tissue. When incorporated into a solid fibrin thrombus, however, plasma fibronectin may fail to bind S. aureus, because the S. aureus-binding sites on fibronectin may be occupied by fibrin. Both S. aureus and fibrin bind to the same 27-kilodalton amino-terminal fragment of fibronectin. To determine whether fibronectin incorporated into fibrin still promotes the adherence of S. aureus, we clotted citrated normal plasma and fibronectin-depleted plasma onto petri dishes. We then measured bacterial adherence to these in vitro fibrin thrombi. We found that the adherence of five of seven S. aureus strains decreased significantly (by 26 to 58%) when fibronectin had been depleted from the fibrin thrombi. Adding fibronectin back reversed this decrease in adherence. The reversal was dose dependent; the increase was in proportion to the amount of fibronectin added back to the plasma. Bacteria known not to bind to fibronectin (Escherichia coli and Staphylococcus epidermidis) adhered 100-fold less than S. aureus, and their adherence was unaffected by the absence of fibronectin in the fibrin thrombus. We conclude that fibronectin incorporated into solid fibrin thrombi does mediate the adherence of most S. aureus strains to fibrin thrombi. Fibronectin may be an important molecule that mediates the adherence of S. aureus to fibrin in wounds.