Cultured human endothelial cells derived from the umbilical cord vein are able to release factor VIII-related antigen into the culture medium. The experiments described in this paper show the presence of two pathways for the secretion of factor VIII-related antigen from endothelial cells. There is a basal release of this antigen, independent of the presence of extracellular calcium ions. This release can be inhibited by cycloheximide and is therefore directly related to de novo protein synthesis. Besides this basal release, there is an extra release of factor VIII-related antigen that can be stimulated by thrombin, the Ca2+-ionophore A23187 or phorbol myristate acetate. As demonstrated by immunofluorescence, the stimulus-inducible release originates from storage granules in the cells. This stimulus-inducible release is dependent on extracellular Ca2+ but independent of intracellular cAMP.