Alcohol consumption during adolescence has long-term sexually dimorphic effects on anxiety behavior and mood disorders. We have previously shown that repeated binge-pattern alcohol exposure increased the expression of two critical central regulators of stress and anxiety, corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP), in adolescent male rats. By contrast, there was no effect of alcohol on these same genes in adolescent females. Therefore, we tested the hypothesis that 17β-estradiol (E2), the predominant sex steroid hormone in females, prevents alcohol-induced changes in CRH and AVP gene expression in the paraventricular nucleus (PVN) of the hypothalamus. To test this hypothesis, postnatal day (PND) 26 females were ovariectomized and given E2 replacement or cholesterol as a control. Next, they were given an alcohol exposure paradigm of 1) saline alone, 2) acute (single dose) or 3) a repeated binge-pattern. Our results showed that acute and repeated binge-pattern alcohol treatment increased plasma ACTH and CORT levels in both E2- and Ch-treated groups, however habituation to repeated binge-pattern alcohol exposure was evident only in E2-treated animals. Further, repeated binge-pattern alcohol exposure significantly decreased CRH and AVP mRNA in Ch-, but not E2-treated animals, which was consistent with our previous observations in gonad intact females. We further tested the effects of E2 and alcohol treatment on the activity of the wild type CRH promoter in a PVN-derived neuronal cell line. Alcohol increased CRH promoter activity in these cells and concomitant treatment with E2 completely abolished the effect. Together our data suggest that E2 regulates the reactivity of the HPA axis to a repeated stressor through modulation of the habituation response and further serves to maintain normal steady state mRNA levels of CRH and AVP in the PVN in response to a repeated alcohol stressor.