Prolonged myocardial stretch typically leads to hypertrophy of cardiomyocytes. As integrins are cellular receptors of stretch, we hypothesize that integrin stimulation induces cardiomyocyte hypertrophy. Integrins of neonatal rat cardiomyocytes (NRCMs) were stimulated with a peptide containing the Arg-Gly-Asp (RGD) sequence for 24 h. For comparison, alpha(1)-adrenergic stimulation by phenylephrine (PE) for 24 h was applied. Saline-treated NRCMs were used as control. The hypertrophic response was quantified by measuring cell surface area (CSA). Phosphorylation of NO-synthase-1 (NOS1) was assessed by immunocytochemistry. CSA was increased by 38% (IQR 31-44%) with RGD and by 68% (IQR 64-84%) with PE versus control (both P < 0.001). NOS-1 phosphorylation was increased by 61% with RGD and by 21% with PE versus control (both P < 0.01). A general NOS-inhibitor (L-NAME) inhibited RGD-induced hypertrophy completely, but had no significant effect on PE-induced hypertrophy. Administration of NO-donor to NRCMs co-incubated with RGD + L-NAME partly restored hypertrophy (to 62% of the hypertrophic effect of RGD alone), but had no effect if incubated with PE + L-NAME. Ryanodine and BAPTA-AM inhibited RGD-induced hypertrophy completely but not that induced by PE. Integrin stimulation of NRCMs by RGD leads to hypertrophy, likely by activation of NOS-1. Abrogation of RGD-induced hypertrophic response upon NOS-inhibition and rescue of this hypertrophic effect by NO-donor suggest that integrin stimulation-induced hypertrophy of NRCMs is NO-dependent.