During the neonatal period, the brain is subject to profound alterations in neuronal circuitry due to high levels of synaptogenesis and gliogenesis. In neuroendocrine regions such as the preoptic area-anterior hypothalamus (POA-AH), the site of GnRH perikarya, these changes could affect the maturation of GnRH neurons. Because the GnRH system is developmentally regulated by glutamatergic neurons, we hypothesized that changes in the N-methyl-d-aspartate (NMDA) receptor system begin early in postnatal development, before the onset of puberty, thereby playing a role in establishing the appropriate environment for the subsequent maturation of GnRH neurons. To this end, we determined developmental changes in NMDA receptors, alterations in GnRH gene expression, and the regulation of GnRH neurons by the NMDA receptor system in developing male and female rats. In Exp I, NMDA receptor subunit (NR) 1 mRNA levels in the POA-AH were found to increase significantly (∼5-fold) from E18 through P10 in both males and females. NR2b mRNA increased significantly between P0 and P5 in both males and females. In contrast, NR2a subunit mRNA, which was in very low abundance in both males and females, increased only in males between P10 and P15. In Exp II we determined that GnRH gene expression changes differentially in developing male and female rats, with increases from P0 to P5 in males, and decreases from P5 to P10 in females. This latter effect in females is attributed to a change in GnRH gene transcription because GnRH primary transcript RNA levels paralleled changes in GnRH mRNA levels. In Exp III, we tested effects of treatment with an NMDA receptor analog on GnRH mRNA levels and found that only P5 and P10 male rats responded to NMDA receptor activation with an increase in GnRH mRNA levels, via a posttranscriptional mechanism. This greater responsiveness of males to NMDA receptor stimulation may be due to differences in the composition and levels of NMDA receptor subunits. Exp IV examined the localization of NR1 in the POA-AH during neonatal development. No GnRH neurons were immunopositive for NR1, indicating that effects of glutamate on GnRH neurons are mediated by interneurons or other glutamate receptor subunits or types. Taken together, these data indicate that glutamatergic inputs to the POA-AH change dramatically during the early postnatal period, before puberty and before the GnRH system is fully responsive to glutamate, consistent with the hypothesis that the maturation of inputs to GnRH neurons, and the establishment of the proper neurotransmitter “milieu” enabling the activation of GnRH neurons, occurs before the onset of puberty.