Fibronectin (FN) plays a role in several adhesion mediated functions including the interaction of platelets with subendothelium. We investigated the role of plasma FN in platelet adhesion and platelet thrombus formation under flow conditions. We used two different perfusion models: the annular chamber with alpha-chymotrypsin-treated rabbit vessel segments, and the flat chamber with coverslips coated with fibrillar purified human collagen type III. Perfusates consisted of washed platelets and washed RBCs, suspended in normal or FN-depleted plasma. Perfusions were carried out for ten minutes at shear rates of 300 or 1,300 s-1. Platelet deposition and thrombus dimensions were evaluated morphometrically by a computerized system. We found that depletion of plasma fibronectin significantly reduced the percentage of total coverage surface and percentage of platelet thrombus, at both shear rates studied, and in both perfusion systems (P less than .01) (P less than .01). The dimensions of the platelet thrombi formed in perfusions at high shear rate were also significantly reduced in perfusions carried out with FN depleted plasma (P less than .01). Addition of purified FN to FN-depleted perfusates restored all values to those measured in the control perfusions. These results indicate that plasma FN is required for platelet aggregate and thrombus formation following adhesion under flow conditions.