We previously reported that GnRH gene expression was enhanced by progesterone (P) in the hypothalamus from ovariectomized and estrogen (OVX+E) treated immature rats. Recent studies indicate that excitatory amino acids may play an important role in the regulation of GnRH secretion and gene expression by steroids. Therefore the present study attempts to examine whether excitatory amino acids are involved in the P-induced GnRH gene expression and release in vitro. MK-801, an NMDA receptor antagonist or CNQX, a non-NMDA receptor antagonist, was administered to OVX+E+P-treated prepubertal female rats. GnRH mRNA was determined by Northern blot hybridization using 32P-labeled antisense RNA, and GnRH release in vitro from the hypothalamic fragments was monitored by GnRH radioimmunoassay. The administration of MK-801 (0.2 mg/kg) for 2 h significantly reduced the P-induced GnRH gene expression and release, whereas CNQX (0.4 mg/kg) had no effect. These results clearly indicate that excitatory amino acids by way of NMDA receptor are involved in the transsynaptic regulation of GnRH gene expression.