The myocardial stretch-induced increase in intracellular [Ca(2+)] ([Ca(2+)](i)) is considered to be caused by integrin stimulation. Myocardial stretch is also associated with increased nitric oxide (NO) formation. We hypothesised that NO is implicated in calcium signalling following integrin stimulation. Integrins of neonatal rat cardiomyocytes were stimulated with a pentapeptide containing the Arg-Gly-Asp (RGD) sequence. [Ca(2+)](i) was measured with Fura2, [NO](i) was measured with DAF2 and phosphorylation of focal adhesion kinase (FAK) was monitored with immunofluorescence techniques. Integrin stimulation increased both [NO](i) and [Ca(2+)](i), the latter response being inhibited by ryanodine receptor-2 (RyR2) blockers and by N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of NOS, but resistant to GdCl(3), diltiazem and wortmannin. Integrin-induced intracellular Ca(2+) release thus appears to be independent of the influx of extracellular Ca(2+) and phosphatidylinositol-3 kinase activity. In addition, integrin stimulation induced phosphorylation of FAK. Our results provide evidence for an integrin-induced Ca(2+) release from RyR2 which is mediated by NO formation, probably via FAK-induced NOS activation.