The D3 domain of von Willebrand factor (VWF) is involved in the multimerization process of the protein through the formation of disulfide bridges. We identified heterozygous substitutions, C1157F and C1234W, in the VWF D3 domain in two unrelated families with unclassified and type 2A von Willebrand disease, respectively. VWF was characterized by a low plasmatic level, an abnormal binding to platelet GPIb and a high capacity of secretion from endothelial cells following DDAVP infusion. Using site-directed mutagenesis and expression in mammalian cells, we have investigated the impact of these mutations upon the multimerization, secretion and storage of VWF. Using COS-7 cells both mutated recombinant VWF (rVWF) displayed only lower molecular weight multimers. Pulse-chase analysis and endoglycosidase H digestion experiments showed the intracellular retention of mutated rVWF in pre-Golgi compartments. Study of hybrid rVWF obtained with a constant amount of wild-type (WT) DNA and increasing proportions of mutated plasmids established that both substitutions reduced the release of WT VWF in a dose-dependent manner and failed to form high molecular weight multimers. Using transfected AtT-20 stable cell lines, we observed similar granular storage of the two mutants and WT rVWF. Our data suggest that cysteines 1157 and 1234 play a crucial role in the early step of the folding of the molecule required for a normal transport pathway, maturation and constitutive secretion. In contrast, their substitution does not prevent the storage and inducible secretion of VWF.