Human endothelial cells express urotensin-II (U-II) as well as its receptor GPR14. Using microfluorimetric techniques, the effect of human U-II on cytosolic Ca2+ concentrations [Ca2+]i in cultured human aortic endothelial cells (HAECs) loaded with Fura-2 was evaluated in static or flow conditions. Under the static state, U-II (100 nM) abolished spontaneous Ca2+ oscillations, which occurred in a population of cultured HAEC. Similarly, U-II reduced thrombin-, but not ATP-induced calcium responses, suggesting that the peptide does not alter the Gq/11/IP3 pathway; rather, it modifies the coupling between protease-activated receptors and Gq/11/IP3. Under the flow condition, U-II (1, 10 and 100 nM) produced a dose-dependent increase in [Ca2+]i, which was subjected to desensitization. The result demonstrates a state-dependent effect of U-II in cultured HAEC, which may explain the variable responses to U-II under different experimental conditions.