The estrogenicity profile of domestic sewage during treatment at a medium-sized (3800 EP) advanced biological nutrient removal plant in Queensland, Australia, was characterized using a sheep estrogen receptor binding assay (ERBA) and the MCF-7 breast cancer cell proliferation assay (E-Screen). The raw influent was highly estrogenic (20-54 ng/L EEq), and primary treatment resulted in a slight increase in estrogenicity that was detected in one of the assays (6-80 ng/L). Concurrent chemical analysis suggested that most of the estrogenicity in the influent was due to natural hormones (>48%). Secondary activated sludge treatment followed by nitrification/denitrification effectively removed > 95% of the estrogenic activity (to <0.75-2.6 ng/L), and estrogenicity of the final tertiary-treated effluent was below the detection limit of both assays (<0.75 ng/L).