Expression of the enkephalin gene in ventromedial hypothalamus (VMH) of the female rat has been correlated with the performance of lordosis behavior. By antisense DNA evidence, it has been drawn into a causal role as well. Here, we explored whether, parallel to earlier molecular and behavioral results, thyroid hormone coadministration could disrupt the estrogenic induction of preproenkephalin (PPE) mRNA. As expected, estradiol benzoate treatment to ovariectomized rats led to a large and significant increase in PPE gene expression in the VMH. This increase was inhibited by coadministration of thyroid hormone. The thyroid hormone interference in PPE gene expression was specific to the VMH, as there were no significant effects in the central nucleus of the amygdala or in the caudate/putamen. These in situ hybridization histochemical results form a direct parallel both to previous transcriptional measurements and to reproductive behavior assays in which thyroid hormones were able to oppose estrogenic facilitation. Previous evidence supports the notion of competitive DNA binding and protein/protein interactions providing mechanisms for nuclear thyroid hormone receptors to affect estrogen receptor function, but other, additional mechanisms cannot be ruled out. To date, both oxytocin and PPE gene expression represent potential hypothalamic systems by which thyroid hormones could interfere with estrogen-stimulated female rat reproductive behavior.