The involvement of excitatory N-methyl-D-aspartate (NMDA) receptors in the hypothalamic control of pituitary LH secretion is well recognized. Recent evidence shows that nitric oxide (NO), a free radical gas, may act as neurotransmitter in the brain, and its efflux is stimulated by activation of NMDA receptors. Studies were undertaken to determine whether NO is involved in the hypothalamic release of LHRH and in the LH surge induced by progesterone (P) in estrogen-primed ovariectomized rats. Rats were ovariectomized and 2 weeks later received estradiol benzoate (30 micrograms sc) at 1000 h. Two days later, P was injected at 1000 h to potentiate the estradiol benzoate-induced LH surge in the afternoon. Serial blood samples were collected at hourly intervals from 1400-1800 h via an intraatrial cannula implanted the day before P injection. Additionally, at various times before onset of the LH surge at 1400 h, the rats were injected sc with one of three inhibitors of NO synthase, the enzyme that generates NO. Control, saline-injected rats showed unambiguous LH surges in the afternoon. However, either a single injection at 1000 h of NG-methyl-L-arginine (20 mg/kg) or three injections at 1000, 1200, and 1400 h of either Nw-nitro-L-arginine methyl ester (NAME, 40 mg/kg) or Nw-nitro-L-arginine (60 mg/kg) to inhibit NO efflux markedly suppressed the P-induced LH surge in the afternoon. To ascertain whether suppression of LH surge was due to blockade of hypothalamic LHRH release, a series of in vitro studies were performed in steroid-primed rats. First we examined the effects of sodium nitroprusside (NPS), a compound that spontaneously generates and releases NO. NPS increased basal and KCl-induced LHRH release in vitro from the medial basal hypothalamus-preoptic area and median eminence fragments. No direct effect of NO at the pituitary level was seen, since NPS did not alter basal or LHRH-induced LH in vitro release from hemipituitaries. In addition, we tested the effects of NAME on NMDA-induced LHRH release in vitro from the median eminence-arcuate nucleus fragments. As expected, NMDA alone (50 mM) induced a significant increase in LHRH release. Addition of NO synthase inhibitor, NAME (1 or 10 mM) to suppress NO efflux, significantly diminished the NMDA-induced LHRH release.