Plasma von Willebrand factor (vWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury. Only the very large vWF multimers are effective in promoting platelet adhesion in flowing blood. A protein disulfide bond reductase in plasma reduces the average multimer size of vWF secreted by endothelial cells. This activity has been isolated from human endothelial cell conditioned medium and shown to be the trimeric glycoprotein, thrombospondin-1 (TSP-1). Incubation of purified TSP-1 with vWF resulted in formation of thiol-dependent complexes of TSP-1 and vWF, generation of new thiols in vWF, and reduction in the average multimer size of vWF. The ratio of the concentrations of TSP-1 and vWF in plasma reflected with average multimer size of vWF. The higher the plasma TSP-1/vWF molar ratio, the smaller the average vWF multimer size. In addition, administration of TSP-1 to mice resulted in reduction in the average multimer size of plasma vWF. Interaction of TSP-1 with vWF is mediated by TSP-1 type 1 properdin domains and the vWF A3 domain. These results indicate that TSP-1 regulates the multimeric size and therefore hemostatic activity of vWF.