Previous experiments have demonstrated that leptin releases luteinizing hormone-releasing hormone (LHRH) from median eminence (ME)-arcuate explants from male rats and also stimulates the release of follicle-stimulating hormone (FSH) and LH from anterior pituitaries with a potency not significantly different from that of LHRH itself. To determine the mechanism by which leptin acts at both the hypothalamic and pituitary level, we evaluated the effect of a competitive inhibitor of nitric oxide synthase (NOS), N^G-monomethyl-L-arginine (NMMA) on the response to leptin. To evaluate the role of NO in the action of leptin to release LHRH, ME-arc explants were incubated with leptin (10⁻¹¹ M), a concentration shown earlier to give the most effective stimulation of LHRH release. NMMA (3 × 10⁻⁴ M) completely inhibited the LHRH release induced by leptin. In other experiments, hemi-anterior pituitaries were incubated with NMMA with and without leptin at various concentrations (10⁻⁹–10⁻⁶ M). As in the case of hypothalamic explants, NMMA had no effect on basal release of LH; however, it completely blocked the stimulation of LH release induced by leptin. Interestingly, the release of LH induced by LHRH (4 × 10⁻⁹ M) was also completely blocked by the inhibitor of NOS. The results provide evidence that leptin acts both at hypothalamic and pituitary level to stimulate NO release, presumably by acting on its receptors at both sites which then induces the release of either LHRH or LH, respectively. Furthermore, LH release induced by LHRH is also mediated by NO.