ABSTRACT Leptin injection increases plasma levels of nitrites-nitrates, an index of nitric oxide (NO) production. As plasmatic levels of NO are correlated with fat mass and since adipose tissue is the main source of leptin, it seems that adipose tissue plays a major role in NO release induced by leptin. Adipocytes express both leptin receptors and nitric oxide synthases (the endothelial isoform: NOS III and the inducible isoform: NOS II). In this study, we demonstrate that physiological concentrations of leptin stimulate NOS activity in adipocytes. This effect of leptin is abolished by i) AG490, an inhibitor of Janus tyrosine kinase-2 (JAK2) / signal transducers and activators of transcription-3 (STAT3), ii) U0126, an inhibitor of mitogen activated protein kinase kinase (MEK) / extracellular signal-regulated kinase (p42/p44 MAPK) and iii) H89 or Rp cAMPS, two inhibitors of protein kinase A, but not by wortmannin, an inhibitor of phosphatidylinositol-3 kinase. Immunoblotting studies show that leptin fails to activate Akt but increases p42/p44 MAPK phosphorylation, an effect which is prevented by U0126 but not by H89. Furthermore, leptin induces NOS III phosphorylation at Ser¹¹⁷⁹ and Thr⁴⁹⁷ but not when adipocytes are pretreated with H89 or U0126. Finally stimulation of adipocyte NOS activity by leptin is either unaltered when protein phosphatase-2A (PP-2A) is inhibited by 1nM okadaic acid or completely abolished when protein phosphatase-1 (PP-1) activity is inhibited by 3 nM tautomycin, which supports a crucial role of PP-1 in mediating this effect of leptin. On the whole, these experiments demonstrate that NOS activity is a novel target for leptin in adipocytes and that the leptin-induced NOS activity is, at least in part, the result of NOS III phosphorylations via both protein kinase A and p42/p44 MAPK activations. More generally this study also leads to consider NO as a potentially important factor for leptin signaling in adipocytes.