We have established that a recombinant von Willebrand Factor (vWF) mutant (vWFdelpro) that lacks the propolypeptide, in contrast with mature wild-type vWF, with which it is identical in terms of primary amino acid sequence, is not able to form a complex with Factor VIII. Wild-type vWF (flvWF) and vWFdelpro were expressed in AtT-20 cells. Under the culture conditions employed, completely processed multimerized flvWF and dimeric vWFdelpro were secreted into the medium. FlvWF and vWFdelpro were compared for their Factor VIII-binding properties in two distinct assay systems. In a direct binding assay, purified human Factor VIII was shown to bind to flvWF that had been immobilized on the surface of microtitre wells by using an anti-vWF monoclonal antibody. In contrast, Factor VIII did not bind to immobilized vWFdelpro. In a competition assay, fluid-phase flvWF appeared to inhibit efficiently the binding of Factor VIII to immobilized vWF isolated from plasma, whereas vWFdelpro did not influence Factor VIII binding. From these observations, it is argued that the pro-polypeptide serves an essential role in the post-translational processes that lead to the expression of a functional Factor VIII-binding site on the mature vWF subunit.