Neurotensin release from estrogen-responsive neurons in the rostral preoptic area of the female rat may play an important role in triggering preovulatory secretion of gonadotropin-releasing hormone on proestrus. We investigated the possibility of sexually differentiated biosynthesis of neurotensin in the rostral preoptic area, using in situ hybridization histochemistry to detect neurotensin/neuromedin N (NT/N) mRNA in adult male rats and adult female rats at proestrus and the first day of diestrus. In sections through the anteroventral periventricular nucleus (AVPv), the number of labeled cells in proestrous females was four times that in males. Diestrus females exhibited half the number of labeled cells present at proestrus, and there was evidence for a significant correlation between circulating estradiol level and number of labeled cells in the AVPv. In the rostral portion of the medial preoptic nucleus (MPN), two contiguous groups of labeled cells were especially prominent. One group, in the medial half of the MPN, was located closer to the midline in females than in males and displayed greater labeling in males than in females. Furthermore, labeling in the rostral MPN was greater at proestrus than at diestrus. These results indicate that biosynthesis of neurotensin and neuromedin N in the rostral preoptic area may be sexually differentiated and, in the female, may vary across the estrous cycle in parallel with circulating estradiol levels, consistent with the view that neurotensin neurons in this area are involved in the regulation of preovulatory secretion of gonadotropin-releasing hormone. The sex- and region-specific expression of NT/N mRNA in the rostral preoptic area suggests functional heterogeneity of neurotensin neuronal populations in this area and implies complex regulation of NT/N gene expression in the rat brain.