The biosynthesis of von Willebrand Factor (vWF) by vascular endothelial cells involves a complex series of processing steps that includes proteolytic cleavage of a 741-residue propeptide and the assembly of disulfide-linked multimers. Using a model system in which experimentally altered vWF cDNAs are expressed in COS-1 cells, we have shown that the vWF propeptide contains determinants that govern the assembly of vWF multimers. Furthermore, the role of the propeptide (in the assembly process) does not require it to be a contiguous part of the pro-vWF primary structure, since independently expressed propeptide was shown to promote the assembly of mature vWF subunits into multimers. Pulse-chase experiments indicated that the independently expressed propeptide formed a transient association with the mature vWF subunit inside the cell. Thus, it appears that the vWF propeptide segment can act in "trans" to direct the assembly of disulfide-linked vWF multimers.