BACKGROUND: Vascular endothelial growth factor (VEGF) and histamine induce von Willebrand factor (VWF) release from vascular endothelial cells. Protein kinase C (PKC) is involved in the control of exocytosis in many secretory cell types. OBJECTIVES: We investigated the role of PKC and the interactions between PKC and Ca2+ signaling in both VEGF-induced and histamine-induced VWF secretion from human umbilical vein endothelial cells (HUVECs). RESULTS: Several PKC inhibitors (staurosporine, Ro31-8220, myristoylated PKC peptide inhibitor and Go6983) block VEGF-induced but not histamine-induced VWF secretion. PKC-alpha and novel PKCs (PKC-delta, PKC-epsilon, and PKC-eta), but not PKC-beta, are expressed in HUVECs. Both VEGF and histamine activate PKC-delta. However, gene inactivation experiments using small interfering RNA indicate that PKC-delta (but not PKC-alpha) is involved in the regulation of VEGF-induced but not histamine-induced secretion. Both VEGF and histamine induce a rise in cytosolic free Ca2+ ([Ca2+]c), but the response to VEGF is weaker and even absent in a significant subset of cells. Furthermore, VEGF-induced secretion is largely preserved when the rise in [Ca2+]c is prevented by BAPTA-AM. CONCLUSIONS: Our study identifies striking agonist specificities in signal-secretion coupling. Histamine-induced secretion is dependent on [Ca2+]c but not PKC, whereas VEGF-induced secretion is largely dependent on PKC-delta and significantly less on [Ca2+]c. Our data firmly establish the key role of PKC-delta in VEGF-induced VWF release, but suggest that a third, VEGF-specific, signaling intermediate is required as a PKC-delta coactivator.