Gonadotropin-releasing hormone (GnRH) neurons represent the primary neuroendocrine link between the brain and the reproductive system. Although they play a key role in stimulating the release of LH and FSH from the anterior pituitary gland, the underlying mechanism by which they trigger the onset of puberty is unclear. To address this issue, RT-PCR, in situ hybridization histochemistry, and Affymetrix gene arrays were used to profile hypothalamic GnRH gene expression in prepubertal and adult rhesus macaques. Like humans, these primates express two molecular forms of GnRH (GnRH-I and GnRH-II), both of which are highly effective at stimulating gonadotropin release via the same GnRHR1 receptor but only GnRH-II showing increased expression in the presence of elevated estrogen concentrations (i.e., positive feedback). Overall, the hypothalamic expression levels of GnRH-I and GnRHR1 were found to be no different between prepubertal and adult animals, despite marked differences in circulating sex-steroid hormone levels. In contrast, the hypothalamic expression level of GnRH-II was significantly higher in the adults. Therefore, although GnRH-I neurons are likely to play a fundamental role in re-initiating LH and FSH release at the end of the juvenile period, in humans and nonhuman primates, it is plausible that the GnRH-II neurons play a key role in maintaining elevated gonadotropin release during the later stages of pubertal development (i.e., at a time when the GnRH-I neurons are subjected to increased negative sex-steroid feedback from the maturing gonads).