von Willebrand factor (vWF) and tissue-type plasminogen activator (tPA) are products of endothelial cells which are secreted into the bloodstream upon a stimulus-induced rise in intracellular Ca(2+). Although the release of both factors appears to be regulated similarly, they exhibit opposing physiological effects in the vasculature with vWF inducing coagulation and platelet aggregation and tPA triggering fibrinolysis and thrombolysis. To analyze possible differences in the regulated secretion of vWF and tPA in more detail, we recorded the Ca(2+)-triggered exocytosis of both factors in cultured human endothelial cells. We demonstrate that vWF and tPA which are stored in different granules within endothelial cells are released with different kinetics following endothelial stimulation with histamine or the Ca(2+) ionophore A23187. While the stimulus-induced release of vWF increases with time over a course of 30 min, maximal acute secretion of tPA is observed 5 min following stimulation and subsequently drops to background levels. In the case of vWF, secretion can also be monitored indirectly through an antibody-reinternalization assay which indicates an incomplete release of vWF during single exocytotic fusion events. Our data thus point to differences in the Ca(2+)-triggered secretion of vWF and tPA which could allow a fine-tuning of their release thereby ensuring a balanced physiological action.