Weibel-Palade bodies are endothelial cell-specific organelles, which contain von Willebrand factor (vWF), P-selectin, and several other proteins. Recently, we found that the small GTP-binding protein Ral is present in a subcellular fraction containing Weibel-Palade bodies. In the present study, we investigated whether Ral is involved in the regulated exocytosis of Weibel-Palade bodies. Activation of endothelial cells by thrombin resulted in transient cycling of Ral from its inactive GDP-bound to its active GTP-bound state, which coincided with release of vWF. Ral activation and exocytosis of Weibel-Palade bodies were inhibited by incubation with trifluoperazine, an inhibitor of calmodulin, before thrombin stimulation. Functional involvement of Ral in exocytosis was further investigated by the expression of constitutively active and dominant-negative Ral variants in primary endothelial cells. Introduction of active Ral G23V resulted in the disappearance of Weibel-Palade bodies from endothelial cells. In contrast, the expression of the dominant-negative Ral S28N did not affect the amount of Weibel-Palade bodies in transfected cells. These results indicate that Ral is involved in regulated exocytosis of Weibel-Palade bodies by endothelial cells.