Urocortin II (UcnII) exerts positive inotropic and lusitropic effects in cardiac myocytes in part via CRF2 receptor-mediated elevations of cAMP. Here, we tested the hypothesis that UcnII also activates the PI3K-Akt pathway to stimulate NO production and modulate contractility via NO-cGMP signalling. In isolated rabbit ventricular myocytes, UcnII (0.1 mM) caused a time-dependent increase in Akt phosphorylation that was maximal after 30 min (+134±28%, n=6, P<0.01 versus time-matched controls; measured using antibodies against phosphorylated and total Akt) and significantly reduced by 0.3 mM wortmannin (n=5) or 10 mM LY294002 (n=5), two structurally different inhibitors of PI3K. With a similar time course, UcnII also increased NO production (+25±7%, n=9, P<0.01; measured by confocal NO imaging using DAF-FM) and fractional shortening (from 12.9±1.0% to 18.0±1.1%, n=9, P<0.01; measured by edge detection). The latter effect was abolished or attenuated by inhibition of guanylyl cyclase (10 mM ODQ, n=7; 10 mM LY83583, n=5) or protein kinase G (0.1 mM KT5823, n=5). The results indicate that, in rabbit ventricular myocytes, UcnII elevates NO production via PI3K-Akt signalling. NO stimulates guanylyl cyclase and protein kinase G to contribute to the increase in contractility.