A bioassay-directed chemical analysis (BDCA) scheme has been developed which combines a yeast screen for estrogenic activity with LC-MS detection after liquid-liquid extraction and fractionation by size exclusion chromatography. Focusing on sewage-treatment plant (STP) effluents, the approach aims at characterizing the substances responsible for estrogenic effects in aquatic systems. Initial results show a strong response of STP effluent extracts in the yeast screen. Estrone, bisphenol A, and nonylphenol have been identified as substances being partly responsible for observed estrogenic activity. However, confirmation experiments with synthetic samples revealed that the estrogenic effect potentials of the samples could not be completely assigned to specific compounds. Further improvement of the limits of detection of the analytical scheme is needed to enable identification and quantification of potent estrogenic compounds at low concentrations.