The estrogenic activity of municipal wastewater in aerated lagoon treatment facilities was evaluated using plasma concentrations of vitellogenin (Vtg) in male fathead minnows (Pimephales promelas). Caged fathead minnows were exposed for 10 to 12 d in three lagoons that are connected in series at each of 10 municipal wastewater treatment facilities in central Iowa. USA, during October and November 2000. Fathead minnows held in the laboratory served as unexposed controls. Pooled (n = 4–10 fish) plasma Vtg, quantified by enzyme‐linked immunosorbent assay (ELISA), was 1,702 ± 670 (mean ± standard error [SE]) μg/ml in the first lagoons (n = 9), 0.94 ± 0.36 μg/ml in the second lagoons (n = 10), and 0.04 ± 0.02 μg/ml in the third lagoons (n = 8). Differences in mean fish plasma Vtg concentration among lagoons were highly significant (p < 0.001). The mean concentration of plasma Vtg in fish in the third lagoons was not significantly different (p = 0.990) from that of the control fish (0.04 ± 0.02 μg/ml). Plasma Vtg concentrations of fish in the first lagoons were inversely correlated with wastewater retention time in the lagoons (p = 0.002, r = −0.877). Water temperatures of the final effluents during the study ranged from 9 to 12°C General treatment efficiency of lagoons has been shown to be dependent on temperature, so the potential exists for decreased removal of estrogenic activity when water temperatures are lower (e.g., winter months) than the present study. In conclusion, wastewater entering aerated lagoon systems was estrogenic to fish, but with serial passage through the lagoons, the estrogenic activity decreased to a level that was not sufficient to induce vitellogenesis in male fathead minnows in a 10‐ to 12‐d exposure.