GnRH neurons in the preoptic area and hypothalamus control the secretion of GnRH and form the final common pathway for hypothalamic-pituitary-gonadal axis regulation in vertebrates. Temporal regulation of reproduction by coordinating endogenous physiological conditions and behaviors is important for successful reproduction. Here, we examined the temporal regulation of reproduction by measuring time-of-day-dependent changes in the electrical activity of GnRH1 neurons and in levels of expression of pituitary gonadotropin mRNA using a daily spawning teleost, medaka (Oryzias latipes). First, we performed on-cell patch-clamp recordings from GnRH1 neurons that directly project to the pituitary, using gnrh1-green fluorescent protein transgenic medaka. The spontaneous firing activity of GnRH1 neurons showed time-of-day-dependent changes: overall, the firing activity in the afternoon was higher than in the morning. Next, we examined the daily changes in the pituitary gonadotropin transcription level. The expression levels of lhb and fshb mRNA also showed changes related to time of day, peaking during the lights-off period. Finally, we analyzed effects of GnRH on the pituitary. We demonstrated that incubation of isolated pituitary with GnRH increases lhb mRNA transcription several hours after GnRH stimulation, unlike the well-known immediate LH releasing effect of GnRH. From these results, we propose a working hypothesis concerning the temporal regulation of the ovulatory cycle in the brain and pituitary of female medaka.