Studies were undertaken to determine whether nitric oxide (NO) is involved in induction of the prolactin surge on proestrus and in that induced by ovarian steroids in ovariectomized (ovx) rats, by using inhibitors of NO synthase, the enzyme that generates NO. Two week-ovariectomized rats were treated either with estradiol benzoate (EB, 30 μg/rat, s.c.) alone, or with EB and 2 days later with progesterone (P, 2 mg/rat, s.c.) to evoke a prolactin surge in the afternoon. Injections of the NO synthase inhibitor N^w-nitro-l-arginine methyl ester (40 mg/kg, s.c.) at 10.00, 12.00, and 14.00 h completely suppressed the steroid-induced prolactin surge in the afternoon. Similarly, another NO synthase inhibitor, N^w-nitro-l-arginine (100 mg/kg, s.c.), injected at 1000, 1200, and 1400 h on proestures suppressed prolactin and luteinizing hormone (LH) surges, but failed to alter the daily increase of corticosterone. These studies confirm our earlier report and show that NO is involved in the complex processes that initiate the afternoon prolactin and LH surges of proestrus.