Subcellular localization of nitric oxide (NO) synthases with effector molecules is an important regulatory mechanism for NO signalling. In the heart, NO inhibits L-type Ca²⁺ channels but stimulates sarcoplasmic reticulum (SR) Ca²⁺ release^,,, leading to variable effects on myocardial contractility. Here we show that spatial confinement of specific NO synthase isoforms regulates this process. Endothelial NO synthase (NOS3) localizes to caveolae^,,, where compartmentalization with β-adrenergic receptors and L-type Ca²⁺ channels allows NO to inhibit β-adrenergic-induced inotropy^,. Neuronal NO synthase (NOS1), however, is targeted to cardiac SR. NO stimulation of SR Ca²⁺ release via the ryanodine receptor (RyR) in vitro, suggests that NOS1 has an opposite, facilitative effect on contractility. We demonstrate that NOS1-deficient mice have suppressed inotropic response, whereas NOS3-deficient mice have enhanced contractility, owing to corresponding changes in SR Ca²⁺ release. Both NOS1^−/− and NOS3^−/− mice develop age-related hypertrophy, although only NOS3^−/− mice are hypertensive. NOS1/3^−/− double knockout mice have suppressed β-adrenergic responses and an additive phenotype of marked ventricular remodelling. Thus, NOS1 and NOS3 mediate independent, and in some cases opposite, effects on cardiac structure and function.