Enkephalins facilitate female reproductive behavior. Within the limbic system and hypothalamus, estrogen induced the expression of preproenkephalin (PPE) mRNA. Estrogen injection caused a biphasic increase in the PPE mRNA levels within the ventromedial hypothalamic nucleus and posterodorsal medial amygdala. The first peak of PPE mRNA levels occurred within an hour, and the second 24 to 48 h after subcutaneous injection of estrogen. The present studies indicated that the rapid first peak of PPE mRNA expression was stress induced, whereas the second peak was estrogen induced. In the posterodorsal medial amygdala but not in the ventromedial hypothalamic nucleus, the antiestrogen, tamoxifen, did not inhibit the first peak, but blocked the second peak of PPE mRNA expression. Subcutaneous oil injection induced a 1-h peak of PPE mRNA levels but not a 24-h peak. Peak levels of plasma corticosterone were coincident with peak PPE mRNA levels. Adrenalectomy plus a constant, low level of corticosterone eliminated the injection-induced increase of corticosterone levels and the subsequent increase in PPE mRNA expression in the ventromedial hypothalamic nucleus and posterodorsal medial amygdala. The present results indicate that both stress steroids and estrogen positively regulate PPE mRNA levels in the ventromedial hypothalamic nucleus and posterodorsal medial amygdala. These results are consistent with the hypothesis that acute, mild stress may contribute to the activation of circuits that facilitate reproductive behavior in the female.