Nitric oxide (NO) has been implicated in the control of the proestrus luteinizing hormone (LH) surge in the rat but to date no studies have attempted to measure neuronal nitric oxide synthase (nNOS) or NO production on proestrus in the hypothalamus in order to determine if endogenous NO is increased on proestrus afternoon to activate gonadotropin-releasing hormone (GnRH) neurons. To address this deficit in our knowledge, we measured nNOS mRNA and protein levels as well as NOS activity levels in rat preoptic area (POA) and medial basal hypothalamus (MBH) fragments at 12.00, 14.00, 16.00, and 18.00 h of proestrus. Serum LH levels were also assessed to determine whether NOS changes correlate to the LH surge. To determine the specificity of observed changes we also measured mRNA levels for the enzyme heme oxygenase-2, which is responsible for production of another putative gaseous transmitter, carbon monoxide. In all studies a metestrus 12.00 h control group was included since steroid and LH levels would be basal at this time as compared to proestrus. The results revealed that nNOS mRNA and protein levels, as well as NOS activity did not change significantly in the MBH on proestrus. In contrast, nNOS mRNA levels were significantly elevated in the POA at proestrus 12.00 and 14.00 h, as compared to metestrus 12.00 h. Likewise, at the protein and activity level, nNOS protein levels in the POA were significantly elevated on proestrus at 14.00 and 16.00 h, with NOS activity significantly increased at 16.00 h on proestrus. The elevation of nNOS protein and activity levels in the POA occurred at the time of initiation of the LH surge. The elevation of nNOS was specific as mRNA levels for the CO-synthetic enzyme heme oxygenase-2 did not change significantly on proestrus in the POA or MBH. As a whole, the current studies provide new evidence that nNOS is elevated in the POA on proestrus, and thus could play a role in the activation of GnRH neurons to produce the preovulatory LH surge.