Platelet adhesion to purified surface-immobilized fibronectin under flow conditions was investigated. Fibronectin was found to support attachment and spreading of platelets. The extent of platelet spreading depended on the amount of immobilized fibronectin. An antiglycoprotein (anti-GP) IIb/IIIa antibody and an Arg-Gly-Asp (RGD)-containing peptide inhibited adhesion almost completely, whereas antibodies directed against platelet GP Ic/IIa (very late antigen 5) inhibited by 50%. Similar results with the antibodies and the peptide were found in a static system. A comparison of different anticoagulants showed no difference in adhesion using citrate or hirudin. However, unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) as the only anticoagulant or in combination with citrate maximally inhibited adhesion by 80% and 60%, respectively. Preincubation of the immobilized fibronectin with UFH resulted in a maximal inhibition of 90%, whereas preincubation with LMWH had no effect. When we preincubated the surface with heparins of different size, we observed 40% inhibition of adhesion with heparins with an average MW of up to 18 kD, whereas a heparin with an average MW of 21 kD almost completely blocked adhesion. These results indicate that platelet adhesion to fibronectin in flow involves several receptors, is highly RGD-mediated, does not require physiologic levels of divalent cations, and can be inhibited by direct binding of heparin to the fibronectin surface.