We have previously reported that two forms of von Willebrand factor (vWf) exist in cultured human umbilical vein endothelial cells: a high molecular weight (HMW) form that is released and can be proteolytically cleaved into a series of plasma-like multimers, and a non-secreted low molecular weight (LMW) form. In this study, the mode of vWf release and the relationship between the two forms were examined. As determined by two-dimensional analysis as well as by immunoreactivity with an antibody to the propolypeptide, the LMW form of endothelial cell vWf consisted of a 260 kD pro-vWf polypeptide, while the HMW form consisted of a 225 kD mature polypeptide. Only the 260 kD polypeptide was susceptible to digestion with endoglycosidase H. Release of the HMW form into the culture media was accompanied by a decrease in cellular vWf. Treatment of endothelial cells with cycloheximide or tunicamycin caused a decrease in the LMW form but did not affect the secretion of the HMW form. These results suggest that two pools of vWf exist in endothelial cells--a LMW form of pro-vWf in the endoplasmic reticulum and a HMW form of mature vWf in the storage compartment. Released vWf derives only from the storage pool.