Abstract--Reduced activity and expression of endothelial NO synthase (eNOS) is observed in cardiomyocytes from pressure-overloaded hearts with heart failure. The present study was aimed to investigate whether reduced eNOS-derived NO production contributes to the hypertrophic growth and phenotype of these cardiomyocytes. Cultured ventricular cardiomyocytes from adult rats were exposed to N-nitro-L-arginine (L-NNA) to inhibit global NO formation, and cultured cardiomyocytes derived from eNOS-deficient mice were used as a model of genetic knockout of eNOS. Cell growth, formation of oxygen-derived radicals (reactive oxygen species [ROS]), activation of p38 mitogen-activated protein (MAP) kinase phosphorylation, and cytokine expression in cardiomyocytes were investigated. L-NNA caused a concentration-dependent acceleration of the rate of protein synthesis and an increase in cell size. This effect was sensitive to p38 MAP kinase inhibition or antioxidants. L-NNA induced a rapid increase in ROS formation, subsequent activation of p38 MAP kinase, and p38 MAP kinase-dependent increases in the expression of transforming growth factor- and tumor necrosis factor-. Similar changes (increased ROS formation, p38 MAP kinase phosphorylation, and cytokine induction) were also observed in cardiomyocytes derived from eNOS+/+ mice when exposed to L-NNA. Cardiomyocytes from eNOS-/- mice displayed higher p38 MAP kinase phosphorylation and cytokine expression under basal conditions, but neither these 2 parameters nor ROS formation were increased in the presence of L-NNA. In conclusion, our data support the hypothesis that reduced eNOS activity in cardiomyocytes contributes to the onset of myocardial hypertrophy and increased cytokine expression, which are involved in the transition to heart failure.