Thrombin-mediated endothelial-cell release of von Willebrand factor (VWF) and P-selectin functionally links protease-activated receptors (PARs) to thrombosis and inflammation. VWF release can be stimulated by both Ca2+ and cAMP, and, although both VWF and P-selectin are found in Weibel-Palade bodies (WPBs), we found that their release could be differentially regulated. In these studies, human umbilical vein endothelial cells stimulated with cAMP or PAR2-AP led to a delayed release of VWF and significantly less P-selectin release compared with histamine, thrombin, or PAR1-AP. Dose-response studies revealed that PAR2-AP was significantly less efficacious in promoting the release of P-selectin compared with VWF. PAR2-AP-induced robust stimulation of intracellular Ca2+ coupled with a significantly greater inhibitory effect of calcium chelation on release of VWF compared with cell-surface expression of P-selectin, suggests an additional Ca2+-independent pathway involved in release of P-selectin. PAR2-AP failed to increase global cAMP levels; however, inhibition of protein kinase A led to a significant attenuation of PAR2-AP-mediated release of VWF. Confocal microscopy studies revealed that PAR2 and forskolin caused preferential release of a population of Weibel-Palade bodies (WPBs) consisting of only VWF. Thus, WPBs are pharmacologically and morphologically heterogeneous, and distinct granule populations are susceptible to differential regulation.