Excitatory amino acids, particularly glutamate, are thought to be important for the maturation of the brain-pituitary-gonadal axis and the induction of puberty in the rat. We have previously shown that, in the female rat, GnRH neurons preferentially express the KA2 and NMDAR2A receptor subunit mRNAs, but not AMPA or NMDAR1 mRNA. The aim of the present study was to determine whether the onset or rate of KA2 and NMDAR2A receptor expression in GnRH neurons is correlated with the onset of puberty. Dual in situ hybridization using digoxigenin-labeled GnRH cRNA probes and 35S-labeled glutamate receptor subunit probes, followed by autoradiography and image analysis were used to measure the KA2 or NMDAR2A mRNA content in GnRH neurons in 20- to 50-day-old female rats which were sacrificed at 08.00 or 17.00 h. The results show that: (a) the KA2 mRNA content of GnRH neurons and the number of GnRH neurons expressing KA2 mRNA increase progressively in the morning hours between postnatal days 20 and 40; (b) the diurnal pattern of KA2 mRNA levels in GnRH neurons changes between days 40 and 50 from high KA2 levels in the morning hours before day 40 to high KA2 mRNA levels in the afternoon in 45- and 50-day-old animals; (c) while the high levels of KA2 mRNA in GnRH neurons in the morning hours of 20- to 40-day-old animals are paralleled by an overall increase in KA2 expression in the preoptic area, the rise in KA2 mRNA in GnRH neurons in the afternoon of 45- and 50-day-old animals appears to be specific for the GnRH neurons, and (d) no significant differences were detected for the NMDAR2A mRNA content in GnRH neurons among the different age groups and the morning and afternoon values. Since the gradual increase in the KA2 mRNA content in GnRH neurons of animals reaching puberty as well as the reversal of diurnal rhythmicity in KA2 receptor mRNA content of GnRH neurons coincide with the times of vaginal opening and first ovulation, it is suggested that glutamate, acting through KA2 receptors directly on GnRH neurons is, at least in part, an important factor in the excitatory regulation of the postnatal sexual development of the female rat. In contrast, expression of the NMDA-preferring receptor, NMDAR2A, in GnRH neurons appeared to be unchanged during this development.