Nebivolol is a highly selective β1-adrenoceptor blocker with additional vasodilatory properties, which may be due to an endothelial-dependent β3-adrenergic activation of the endothelial nitric oxide synthase (eNOS). β3-adrenergic eNOS activation has been described in human myocardium and is increased in human heart failure. Therefore, this study investigated whether nebivolol may induce an eNOS activation in cardiac tissue. Immunohistochemical stainings were performed using specific antibodies against eNOS translocation and eNOS serine1177 phosphorylation in rat isolated cardiomyocytes, human right atrial tissue (coronary bypass-operation), left ventricular non-failing (donor hearts) and failing myocardium after application of the β-adrenoceptor blockers nebivolol, metoprolol and carvedilol, as well as after application of BRL 37344, a specific β3-adrenoceptor agonist. BRL 37344 (10 μM) significantly increased eNOS activity in all investigated tissues (either via translocation or phosphorylation or both). None of the beta-blockers (each 10 μM), including nebivolol, increased either translocation or phosphorylation in any of the investigated tissues. In human failing myocardium, nebivolol (10 μM) decreased eNOS activity. In conclusion, nebivolol shows a tissue-specific eNOS activation. Nebivolol does not activate the endothelial eNOS in end-stage human heart failure and may thus reduce inhibitory effects of NO on myocardial contractility and on oxidative stress formation. This mode of action may be of advantage when treating heart failure patients.