Recent studies have demonstrated that urotensin-II (U-II) plays important roles in cardiovascular actions including cardiac positive inotropic effects and increasing cardiac output. However, the mechanisms underlying these effects of U-II in cardiomyocytes still remain unknown. We show by electrophysiological studies that U-II dose-dependently potentiates L-type Ca2+ currents (I-Ca,I- L) in adult rat ventricular myocytes. This effect was U-II receptor (U-IIR)-dependent and was associated with a depolarizing shift in the voltage dependence of inactivation. Intracellular application of guanosine-5'-O-(2-thiodiphosphate) and pertussis toxin pretreatment both abolished the stimulatory effects of U-II. Dialysis of cells with the QEHA peptide, but not scrambled peptide SKEE, blocked the U-II-induced response. The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin as well as the class I PI3K antagonist CH132799 blocked the U-II-induced I-Ca,I- L response. Protein kinase C antagonists calphostin C and chelerythrine chloride as well as dialysis of cells with 1,2bis(2aminophenoxy)-ethaneN, N,N',N'-tetraacetic acid abolished the U-II-induced responses, whereas PKC beta inhibition or PKA blockade had no effect. Exposure of ventricular myocytes to U-II markedly increased membrane PKC beta(1) expression, whereas inhibition of PKC beta(1) pharmacologically or by shRNA targeting abolished the U-II-induced I-Ca,I- L response. Functionally, we observed a significant increase in the amplitude of sarcomere shortening induced by U-II; blockade of U-IIR as well as PKC beta inhibition abolished this effect, whereas Bay K8644 mimicked the U-II response. Taken together, our results indicate that U-II potentiates I-Ca,I- L through the beta gamma subunits of G(i/o)-protein and downstream activation of the class I PI3K-dependent PKC beta(1) isoform. This occurred via the activation of U-IIR and contributes to the positive inotropic effect on cardiomyocytes.