Gonadotropin-releasing hormone (GnRH) neurons form the final common pathway for the central regulation of reproduction. Gamma-amino butyric acid (GABA), the main inhibitory neurotransmitter in the adult brain, has long been implicated in playing key roles in the regulation of GnRH neurons. Two groups reported recently that GABA depolarizes GnRH neurons, although one group reported a hyperpolarizing action of GABA. In this study, we investigated the GABA-induced changes in [Ca² ]_i of GnRH neurons from GnRH-enhanced green fluorescent protein (GnRH-EGFP) rats both to confirm the depolarizing action of GABA and to further examine the developmental and estrous cycle-dependent modulations of GABA action. GABA increased [Ca² ]_i in GnRH neurons at all developmental stages of both sexes. GABA also increased [Ca² ]_i in adult female GnRH neurons prepared in the afternoon at each estrous cycle stage. The percentages of neurons with increased [Ca² ]_i were 90% in proestrus, 59% in estrus, 84% in diestrus I, and 89% in diestrus II. In GnRH neurons prepared from adult females in the morning, however, the percentage was significantly lower than in those prepared in the afternoon, except in estrus. The percentage was also lower in adult males than in adult females. GABA responses were mimicked by muscimol and blocked by bicuculline. In addition, removal of extracellular Ca² completely suppressed the GABA action, and bumetanide attenuated the response. These results indicate that GABA depolarizes GnRH neurons by activating GABA_A receptors, thereby activating voltage-gated Ca² channels and facilitating Ca² influx. In addition, the response to GABA is modulated according to the estrous cycle stage, diurnal rhythm, and sex.