The medial preoptic area (MPOA) is a critical regulatory site for the control of male sexual behavior. We first measured glutamate in 2 min microdialysate samples from the MPOA before, during, and after copulation by male rats. There was a slight [approximately 140% of baseline (BL)] rise in extracellular glutamate when the female was presented, a significant increase (approximately 170% of BL) during periods of mounting and intromitting, and a very large increase in samples collected during ejaculation (approximately 300% of BL). A precipitous fall in levels occurred in the first postejaculatory sample; the magnitude of this fall was highly correlated with the length of the postejaculatory interval of quiescence. In experiment 2, we reverse-dialyzed a mixture of glutamate uptake inhibitors into the MPOA before and during mating; control animals received artificial CSF. The mixture increased extracellular glutamate (approximately 280% of BL), increased the number of ejaculations in the 40 min test, decreased ejaculation latency, and decreased the postejaculatory latency to resume copulation. These data, together with other findings that glutamate in the MPOA can elicit genital reflexes in anesthetized rats and that glutamate receptor antagonists in the MPOA impair copulation, strongly suggest that MPOA glutamate is a major facilitator of copulation and that the postejaculatory fall in glutamate regulates the postejaculatory interval.