Cnemidophorus uniparens is a unisexual species of whiptail lizard of hybrid origin whereas C. inornatus is a sexual species and the maternal ancestor of C. uniparens. Together they represent an excellent model system for investigating the evolution of hormone-brain-behavior relationships. Normal circulating estradiol (E) concentrations in C. uniparens are approximately 5-fold lower than those of female C. inornatus in a similar reproductive state. Experiments were performed to determine whether (i) C. uniparens is more sensitive to E, and (ii) whether the difference in sensitivity is correlated with differences in estrogen receptor (ER)-mRNA expression in the brain. Dose-response curves reveal that ovariectomized C. uniparens are more responsive than ovariectomized C. inornatus to exogenous estradiol 17β-benzoate (EB). EB is more effective in C. uniparens at inducing receptive behavior and progesterone receptor (PR) gene expression in the ventromedial nucleus of the hypo-thalamus (VMH). In situ hybridization analysis of ER-mRNA expression revealed no species differences in ER-mRNA content in the VMH of ovariectomized animals. Treatment of ovariectomized animals with EB resulted in a greater induction of ER-mRNA expression in the VMH of C. uniparens compared to C. inornatus. These results indicate that the differences in behavioral sensitivity to E lie in the estrogen target neurons in the brain region controlling receptive behavior, the VMH, and that the difference in sensitivity cannot be explained by species differences in the basal rate of ER gene expression.