Background: Nitric oxide (NO) has emerged as an important neurotransmitter involved in the control of the neuroendocrine function. NO acts at hypothalamic, pituitary, and gonadal levels. Previous data from our laboratory showed that blockade of NO generation, after systemic administration of a NO synthase inhibitor (Nω-nitro-arginine methyl ester, NAME), increased the luteinizing hormone (LH) secretion in intact and ovariectomized females, whereas a blockade of spontaneous and steroid-induced LH and prolactin surges after NO synthase inhibition has been also described. Methods and Results: Adult male rats were implanted with chronic intra-auricular cannulae and 5 days later sampled at 15-min intervals during 6 h (10.00–16.00 h). Administration of NAME (40 mg/kg at 08.00 and 13.00 h) stimulated significantly (p ≤ 0.01) the LH secretion, increasing LH pulse amplitude (0.58 ± 0.14 vs. 0.08 ± 0.01 ng/ml in controls), mean LH levels (0.64 ± 0.15 vs. 0.15 ± 0.03 ng/ml in controls), and area under curve (239 ± 56 vs. 57 ± 13 in controls). This effect was blocked by coadministration of sodium nitroprusside (SNP), a NO donor (0.5 mg/kg). The action of NAME was observed 3 h after administration, in contrast to the earlier response detected in female rats, and it appeared selective for LH, as prolactin and growth hormone secretion remained unchanged. Further analysis was carried out to determine whether the effect of NAME on the LH secretion was indirect and mediated by changes in testosterone release. To this end, adult male rats were decapitated 2 h after administration of NAME (40 mg/kg), SNP (0.5 mg/kg), or L-nitro-arginine methyl ester ( L-AME), a substrate for NOS (1 g/kg). The serum testosterone concentrations were unchanged after NAME administration, but inhibited by SNP and L-AME. Finally, the effect of NAME and SNP on in vitro testosterone secretion was analyzed. NAME (10 m M) did not affect basal testosterone production, but inhibited the human chorionic gonadotropin stimulated testosterone secretion. Conclusions: These data strongly suggest that the stimulatory effect of NAME on LH secretion is not due to an inhibition of testosterone release and is exerted at the hypothalamic-pituitary level.