The modulatory role of endothelial nitric oxide synthase (eNOS) on heart contraction, relaxation and rate is examined in light of recent studies using genetic deletion or overexpression in mice under specific conditions. Unstressed eNOS‐/‐ hearts in basal conditions exhibit a normal inotropic and lusitropic function, with either decreased or unchanged heart rate. Under stimulation with catecholamines, eNOS‐/‐ mice predominantly show a potentiation in their β‐adrenergic inotropic and lusitropic responsiveness. A similar phenotype is observed in β3‐adrenoceptor deficient mice, pointing to a key role of this receptor subtype for eNOS coupling. The effect of eNOS on the muscarinic cholinergic modulation of cardiac function probably operates in conjunction with other NO‐independent mechanisms, the persistence of which may explain the apparent dispensability of this isoform for the effect of acetylcholine in some eNOS‐/‐ mouse strains. eNOS‐/‐ hearts submitted to short term ischaemia‐reperfusion exhibit variable alterations in systolic and diastolic function and infarct size, while those submitted to myocardial infarction present a worsened ventricular remodelling, increased 1 month mortality and loss of benefit from ACE inhibitor or angiotensin II type I receptor antagonist therapy. Although non‐conditional eNOS gene deletion may engender phenotypic adaptations (e.g. ventricular hypertrophy resulting from chronic hypertension, or upregulation of the other NOS isoforms) potentially confounding the interpretation of comparative studies, the use of eNOS‐/‐ mice has undoubtedly advanced (and will probably continue to improve) our understanding of the complex role of eNOS (in conjunction with the other NOSs) in the regulation of cardiac function. The challenge is now to confirm the emerging paradigms in human cardiac physiology and hopefully translate them into therapy.