The aim of the study was to evaluate the possible contribution of non-endothelial eNOS to the regulation of blood pressure (BP). To accomplish this, a double transgenic strain expressing eNOS exclusively in the vascular endothelium (eNOS-Tg/KO) has been generated by endothelial-specific targeting of bovine eNOS in eNOS-deficient mice (eNOS-KO). Expression of eNOS was evaluated in aorta, myocardium, kidney, brain stem and skeletal muscle. Organ bath studies revealed a complete normalization of aortic reactivity to acetylcholine, phenylephrine and the NO-donors in eNOS-Tg/KO. Function of eNOS in resistance arteries was demonstrated by acute i.v. infusion of acetylcholine and the NOS-inhibitor L-NAME. Acetylcholine decreased mean arterial pressure in all strains but eNOS-KO responded significantly less sensitive as compared eNOS-Tg/KO and C57BL/6. Likewise, acute i.v. L-NAME application elevated mean arterial pressure in C57BL/6 and eNOS-Tg/KO, but not in eNOS-KO. In striking contrast to these findings, mean, systolic and diastolic BP in eNOS-Tg/KO remained significantly elevated and was similar to values of eNOS-KO. Chronic oral treatment with L-NAME increased BP to the level of eNOS-KO only in C57BL/6, but had no effect on hypertension in eNOS-KO and eNOS-Tg/KO. Taken together, functional reconstitution of eNOS in the vasculature of eNOS-KO not even partially lowered BP. These data suggest that the activity of eNOS expressed in non-vascular tissue might play a role in physiologic BP regulation.