Nitric oxide (NO) plays a central role in regulating vascular tone. Three types of NO-synthases exist in mammalian cells. The calcium-dependent forms endothelial NOS (eNOS) and neuronal NOS (nNOS) and the constitutively-active inducible NOS (iNOS). Leptin is an adipose tissue-derived peptide hormone which also affects the cardiovascular system as it induces angiogenesis and activates mitogenic processes. In this study, we investigated the effect of leptin on vascular function and NO-production. Wildtype (WT) and eNOS knockout (eNOS-/-) mice were treated with or without leptin (0,5 mg/kg/day by minipump) for 14 days. Isolated vessel recordings revealed that acetylcholine (ACh)-induced relaxation observed in aortic rings from wildtype was absent in rings from eNOS-/- mice. Surprisingly, after in vivo leptin-application, aortic rings from eNOS-/- mice responded to ACh with endothelium-dependent relaxation. Blocking NOS activity by nitro-L-arginine (L-NA) abolished reactivity to ACh in all rings (WT and eNOS-/- with or without leptin infusion). When NO-availability was estimated from L-NA-induced constrictions in endothelium-intact rings, NO was undetectable in native rings from eNOS-/-. In contrast, after infusion of leptin, basal NO-production was increased in rings from WT and importantly became detectable in eNOS-/- mice. As eNOS can be excluded as a source of NO and as iNOS activity is constitutive, this leptin-induced endothelium-dependent response is probably mediated by nNOS. Indeed, in WT aortic rings, ex vivo incubation with leptin increased nNOS but not e- and iNOS mRNA expression. In cultured human endothelial cells, leptin increased JAK2 and STAT3 phosphorylation, signals, which are thought to control nNOS expression. We conclude that leptin infusion into mice increases the vascular production of NO by nNOS induction. This process may represent a compensatory mechanism for the vascular dysfunction occuring in obese subjects.