The intracellular signaling of human urotensin II (hU-II) and its interaction with other vasoconstrictors such as angiotensin II are poorly understood. In endothelium-denuded rat aorta, co-administration of hU-II (1 nmol/L) and angiotensin II (2 nmol/L) exerted a significant contractile effect which was associated with increased protein kinase C (PKC) activity as well as phosphorylation of PKC/II and myosin light chain (MLC), whilst either hU-II or angiotensin II administered alone at these concentrations had no statistically significant effect. This synergistic effect was abrogated by either PKC inhibitor chelerythrine (10 µmol/L and 30 µmol/L), selective PKC/II inhibitor Go976 (0.1 µmol/L and 1 µmol/L), hU-II receptor ligand urantide (30 nmol/L and 1 µmol/L), or angiotensin II antagonist Losartan (1 µmol/L). Moreover, in endothelium-intact rat aorta, the synergistic effect of hU-II and Ang II was not exerted any longer and this synergistic effect was unmasked by pretreatment of the nitric-oxide synthase inhibitor N^G-nitro-L-arginine methyl ester. hU-II (10 nmol/L) alone caused a long-lasting increase in phospho-PKC, phospho-MLC, and PKC activity which was associated with the long-lasting vasoconstriction. These changes were prevented by chelerythrine. Methoxyverapamil/thapsigargin treatment reduced the hU-II-induced vasoconstriction by 50%. The methoxyverapamil/thapsigargin-resistant component of hU-II-induced vasoconstriction was dose-dependently inhibited by chelerythrine. In conclusion, hU-II induces a novel PKC-dependent synergistic action with angiotensin II in inducing vasoconstriction. /II is probably the PKC isoform involved in this synergistic action. Nitric oxide produced in the endothelium probably masks this synergistic action. The long-lasting vasoconstriction induced by hU-II alone is PKC-dependent and associated with PKC phosphorylation.