Environmental endocrine disruptors can profoundly impact sex ratios of fish popula- tions, especially if the fish exhibits plasticity in sex determination. The Atlantic silverside Menidia menidia exhibits a genetically based latitudinal cline in numerous traits including temperature- dependent sex determination (TSD), which varies from high thermal influence on sex determination in southern fish, to pure genetic sex determination (GSD), with no thermal influence in northern fish. We used this gradient in TSD/GSD to test for differential population susceptibility to a common municipal wastewater constituent, 17 β-estradiol (E2), among 4 populations with varying levels of TSD. Larvae were exposed to E2 during sex differentiation and assessed for sex ratios. The sex ratio in southern populations was more responsive to E2 than in northern populations, indicating differen- tial population sensitivity. To assess the potential sensitivity of wild populations to estrogenic com- pounds, we measured the sex ratio in 12 M. menidia populations over a 3 yr period along a pro- nounced longitudinal gradient in sewage effluent and urbanization across Long Island. Water temperatures were measured to account for thermal influence on sex ratio. We found a significant trend toward female-biased sex ratios in more urbanized estuaries, with lower proportions of females in less impacted estuaries. Temperature was ruled out as the primary cause. Because sewage efflu- ent often carries high concentrations of estrogenic compounds, and our experiments confirmed a heightened sensitivity to E2 in fish with TSD, the most likely reason for the longitudinal trend in sex ratio is a gradient in the concentration of estrogenic compounds associated with urbanization.