We have used monoclonal antibodies (M Abs) and proteolytic fragmentation to localize structurally the functional sites of human von Willebrand factor (vWF) responsible for interaction with membrane glycoproteins GPIb, GPIIb/IIIa, and with collagen. SpII (215 kd) and SpIII (320 kd), the S aureus V-8 protease homodimeric fragments representing the carboxy-terminal and amino-terminal segments of the vWF subunit, competitively inhibited the binding of multimeric vWF to thrombin-stimulated or ristocetin-stimulated platelets, respectively. Specific saturable binding of each fragment was observed to stimulate platelets appropriately and was inhibited only by selected M Abs that both bound to the specific fragment and inhibited the corresponding function. M Ab 9, which blocks thrombin-induced binding of vWF to platelets, inhibited binding of SpII to platelets and bound to SpII as well as to a dimeric, 86-kd thermolysin fragment composed of 42-kd and 23-kd subunits, each possessing the epitope. Binding of SpII was also inhibited by a M Ab to GPIIb/IIIa. Thus, it appears that a portion of the carboxy-terminal end of vWF contains the ligand site for the GPIIb/IIIa receptor. In contrast, M Ab H9, which blocks ristocetin-induced binding of vWF to platelets, inhibited binding of SpIII to platelets and bound to SpIII as well as to monomeric 33-kd and 28-kd subtilisin fragments. Binding of SpIII to platelets was also inhibited by a M Ab to GPIb. Thus, it appears that a small segment of the amino-terminal part of vWF contains the ligand for the platelet GPIb receptor. The collagen binding site of vWF was localized with M Ab B203, which inhibits vWF interaction with collagen. This M Ab also bound to SpIII as well as to monomeric 26-kd and 23-kd subtilisin fragments. Thus, the third functional site responsible for collagen binding appears to be localized on the amino-terminal portion of vWF, in a linear sequence different from those responsible for interaction with either of the platelet receptors. These assignments of functional sites should facilitate the localization of structural defects of vWF in the various forms of vWD and support the role of vWF as an adhesive protein with multiple interactive sites.