Exposure to nitrous oxide (N2O) may result in decreased fertility and other reproductive problems in women working in dental offices. This study explored direct effects of N2O on gonadotropin-releasing hormone (GnRH) neurons involved in pituitary-gonadal function. An immortalized GnRH-secreting GT1-7 cell line was used to evaluate the effects of 60% N2O exposure for 24 hours on pro-GnRH mRNA levels, KCl-stimulated GnRH levels, and certain signaling pathways. N2O exposure for 24 hours decreased pro-GnRH mRNA levels by approximately 80% without affecting cell viability or intracellular GnRH levels. The ability of KCl to release GnRH stores was also significantly reduced. Western blot analyses of ERK1/2, and p38 phosphorylation and IkappaB did not indicate any change in phosphorylation, although p63 tyrosine phosphorylation was increased after short-term N2O exposure. Levels of c-fos, a nuclear transcription factor, were not changed by N2O exposure. These results indicate that N2O significantly decreases evoked release of GnRH as well as GnRH mRNA levels either by decreased synthesis or by mRNA stability. These results may be important in understanding adverse effects of N2O on female workers in dental practice where the gas is not scavenged.