Epidemiological, ecological, and laboratory-based studies support the hypothesis that endocrine disrupting chemicals (EDCs) in the environment are responsible for developmental and reproductive abnormalities. We have previously described a killifish population resident in a highly polluted Superfund site (New Bedford Harbor, NBH) that shows evidence of exposure to an estrogenic environment and endocrine disruption. Here, we compare NBH with a local reference population (Scorton Creek, SC) for developmental patterns and direct effects of exogenous estradiol on the estrogenic markers, brain cytochrome P450 aromatase (CYP19A2 or AroB), hepatic vitellogenin (Vtg), and hepatic estrogen receptor alpha (ER alpha). In contrast to our previous observation of elevated ER alpha in NBH embryos, developmental levels of AroB and Vtg mRNAs did not differ between the two sites, demonstrating that not all estrogen-responsive genes are upregulated in NBH embryos. A dose-response experiment showed that NBH larvae are less responsive (lower maximum induction, as measured by ER alpha) and less sensitive (higher EC(50) for induction, as measured by AroB) to estradiol than SC larvae, changes that would be adaptive in an estrogenic environment. In contrast, induction of Vtg mRNA is similar in the two populations, indicating that the adaptive mechanism is target gene-specific. Based on the lower basal levels of ER alpha mRNA in several tissues from adult NBH fish vs SC fish (Greytak and Callard, 2007), we predicted estrogen hyporesponsiveness; however, induction of ER alpha by estradiol exposure in reproductively inactive males did not differ between the two sites. Moreover, AroB was more responsive and Vtg induction was greater (2d) or similar (5d) in NBH as compared to SC males. Worth noting is the high inter-individual variability in estrogen responses of gene targets, especially in NBH killifish, which may indicate evolving preadaptive or adaptive mechanisms. In conclusion, although multi-generational exposure to a highly polluted environment is associated with changes in basal levels of ER alpha mRNA, this is not a simple predictor of estrogen responsiveness. We hypothesize that adaptation of killifish to the estrogenic and polluted environment may be occurring through diverse mechanisms that are gene-, tissue type- and life-stage-specific.