The binding of human von Willebrand factor (vWF) to a variety of extracellular matrix components immobilized on plates and the binding of vWF to platelet glycoprotein Ib (GPIb) after interacting with these matrix components were examined by means of an enzyme-linked immunosorbent assay. vWF preferably bound to type III collagen, whereas it did not significantly bind to type I, IV, V, or VI collagen, fibronectin, laminin, elastin, or proteoglycans. Soluble type III collagen did not bind to vWF coated on plates and showed a little effect on the vWF binding to the immobilized collagen, suggesting that solid-phase collagen is important for the interaction with vWF. When glycocalicin, the N-terminal carbohydrate-rich extracellular domain of GPIb alpha exhibiting the vWF-binding activity, was added to vWF bound to collagen type III, no significant binding of glycocalicin was observed, but it bound to vWF in the presence of botrocetin, a vWF modulator protein isolated from Bothrops jararaca snake venom. These results indicate that vWF immobilized on collagen can interact with GPIb but that binding of vWF to the collagen matrix alone is insufficient for modulating vWF so that it interacts with GPIb under static conditions. Another unknown physiological modulator functionally mimicking botrocetin or high-shear stress may be involved in the platelet adhesion to extracellular matrix in the early stage of hemostasis.