Abstract-The seasonal variations of estrogenic compounds and the estrogenicities of influent and effluent were investigated by chemical analysis and in vitro assay in a municipal sewage treatment plant in Wuhan (China). The levels of eight estrogenic compounds, including 17-estradiol (E 2 ), estrone (E 1 ), estriol (E 3 ), diethylstilbestrol (DES), 17␣-ethinylestradiol, nonylphenol (NP), 4-tert-octylphenol (OP), and bisphenol A (BPA), were measured by gas chromatography-mass spectrometry. Total estrogenic activity of sewage was quantitatively assessed using primary cultured hepatocytes of male Megalobrama amblycephala Yih using vitellogenin as a biomarker. The E 2 equivalents (EEQs) obtained from the chemical analysis were consistent with those measured by bioassay. The natural (E 1 , E 2 , and E 3 ) and synthetic (DES) estrogens, as well as NP, were the main contributors of the total EEQs of influent and effluent in the present study. The levels of natural estrogens E 1 and E 3 in the influent and effluent were higher in winter than in summer, whereas the situation for NP and OP was the reverse. The levels of E 2 , DES, and BPA varied little among different seasons. 17␣-Ethinylestradiol was not detected in the influent and effluent. The estrogenicities of the influent and of the primary and secondary effluents were all higher in summer than in winter. Estrogenic activities in winter mainly originated from natural (E 1 , E 2 , and E 3 ) and synthetic (DES) estrogens, whereas the increase of EEQs in summer was contributed by NP. The results from chemical analysis and bioassay demonstrate that estrogenic compounds cannot be entirely removed by the existing sewage treatment process, which should be further improved to protect aquatic ecosystems and human health.