In situ hybridization techniques were used in the present study to detect hypothalamic expression of the oxytocin receptor (OR) gene. Binding studies have localized OR to various brain regions and have detected a high density of receptors in the ventromedial hypothalamus (VMH), a documented target of estrogen action. This study was designed to compare levels of OR messenger RNA (mRNA) in the VMH of male and female rats and to study the effects of estrogen treatment on mRNA levels in the VMH of female rats. After cloning a rat OR gene from a genomic testes library, a probe was generated for use in in situ hybridization assays to evaluate sex differences in OR mRNA expression in the VMH. In addition, ovariectomized females were treated with estrogen, and VMH OR mRNA expression was compared with that in ovariectomized or intact females. The results of these studies showed that male rats expressed higher levels of OR mRNA in the VMH than females. Estrogen-treated ovariectomized females exhibited significantly greater expression in the VMH than either oil-treated or intact females. These results support binding studies that have shown oxytocin binding in the VMH to be regulated by gonadal steroids and suggest that estrogen may either directly or indirectly regulate transcription of the OR gene.