NO released by endothelial cells indirectly regulates cardiac function through modulation of the coronary reserve. In addition, nitric oxide synthases expressed in different subcellular compartments in cardiac myocytes regulate distinct aspects of excitation-contraction coupling. We identified the endothelial isoform of nitric oxide synthase (eNOS, encoded by NOS3) in T-tubular caveolae, close to junctional SR. Stretching of cardiac muscle or isolated cardiomyocytes activates the phosphorylation of eNOS on Ser1177 through PI3K/Akt-dependent signaling. This is accompanied with an increase in NO production in cardiomyocytes, as well as increased Ca sparks rate, calcium transients and contraction force. This is clearly dependent on NO production by eNOS, since the stretch-induced increase of Ca release and contraction are abolished by NOS inhibition and absent in cardiomyocytes from mice genetically deficient in eNOS. Therefore, eNOS activation participates in the length-dependent recruitment of contractile reserve in response to stretch. Downregulation of eNOS or uncoupling of the enzyme in the failing heart probably contributes to the degradation of the inotropic state. This would be reversed by pharmacologic treatments that restore eNOS expression/activity in cardiomyocytes, e.g. with beta-adrenergic blockers or statins.