—von Willebrand factor (vWF), an adhesive glycoprotein involved in primary hemostasis, is stored and released from endothelial secretory granules called Weibel-Palade bodies. Regulated secretion occurs in reaction either to [Ca²⁺]_i-raising agents (histamine or thrombin) or to cAMP-raising agents (epinephrine, adenosine, or forskolin). We investigated the pattern of release and the cytoskeletal requirements for secretion in response to these 2 classes of agonists. Secretion induced by [Ca²⁺]_i-raising agents involves peripheral and central granules and is inhibited by colchicine-induced microtubule disruption. It is accompanied by Rho-dependent stress fiber formation and cell retraction. Secretion and remodeling occur in the same individual cells. However, secretion is potentiated by cytochalasin E and C3 toxin, indicating that stress fiber formation antagonizes vWF secretion. In contrast, vWF secretion induced by cAMP-raising agents involves the release of only peripheral granules (implying less vWF release on a per cell basis) and is not inhibited by microtubule disruption. cAMP-mediated secretion is accompanied by disruption of stress fibers, strengthening of the cortical actin rim, and preservation of cell-cell contacts. It is unaffected by cytochalasins or C3 toxin. In contrast to [Ca²⁺]_i-raising agents, cAMP-raising agents induce secretion without cell retraction/intercellular gap formation. Thus, they are likely to play a physiological role in the regulation of endothelial vWF secretion and, therefore, of plasma vWF levels.