NO acting through soluble guanylyl cyclase and cGMP formation is a negative regulator of cardiomyocyte hypertrophy. Downstream targets mediating the inhibitory effects of NO/cGMP on cardiomyocyte hypertrophy have not been elucidated. In addition to its antihypertrophic effects, NO promotes apoptosis in cardiomyocytes, presumably through cGMP-independent pathways. We investigated the role of cGMP-dependent protein kinase (PKG) in the antihypertrophic and proapoptotic effects of NO. Incubation of neonatal rat cardiomyocytes with the NO donorS-nitroso-N-acetyl-d,l-penicillamine (SNAP) (250 &mgr;mol/L) or the PKG-selective cGMP analog 8-pCPT–cGMP (500 &mgr;mol/L) activated endogenous PKG type I, as shown by the site-specific phosphorylation of vasodilator-stimulated phosphoprotein, a well-characterized PKG substrate. SNAP (250 &mgr;mol/L) and 8-pCPT–cGMP (500 &mgr;mol/L) modestly attenuated the hypertrophic response to &agr;1-adrenergic stimulation with phenylephrine. Although a high concentration of SNAP (1000 &mgr;mol/L) promoted apoptosis in cardiomyocytes, as evidenced by the formation of histone-associated DNA fragments, antihypertrophic concentrations of SNAP (250 &mgr;mol/L) and 8-pCPT–cGMP (500 &mgr;mol/L) did not promote cell death. Because chronic activation downregulated endogenous PKG I, we explored whether gene transfer of PKG I would enhance the sensitivity of cardiomyocytes to the antihypertrophic effects of NO/cGMP. Indeed, after adenoviral overexpression of PKG I&bgr;, SNAP (250 &mgr;mol/L) and 8-pCPT–cGMP (500 &mgr;mol/L) completely suppressed the hypertrophic response to &agr;1-adrenergic stimulation. As observed in noninfected cells, SNAP (250 &mgr;mol/L) and 8-pCPT–cGMP (500 &mgr;mol/L) did not promote apoptosis in cardiomyocytes overexpressing PKG I&bgr;. Moreover, overexpression of PKG I&bgr; did not enhance the proapoptotic effects of 1000 &mgr;mol/L SNAP, implying PKG-independent effects of NO on apoptosis. Endogenous PKG I mediates antihypertrophic but not proapoptotic effects of NO in a cell culture model of cardiomyocyte hypertrophy. Adenoviral gene transfer of PKG I selectively enhances the antihypertrophic effects of NO without increasing the susceptibility to apoptosis.