Sediment extracts from a creek in the Neckar river basin (Germany), which received the discharge of treated hospital wastewater, were found to exhibit strong aromatic hydrocarbon (Ah) receptor-mediated effects in a rainbow trout liver cell line (RTL-WI) as well as high mutagenicity in the Salmonella/microsome assay after fractionation. The crude extract did not exhibit a clear mutagenic response. Apparently, cleanup or fractionation before mutagenicity testing is necessary to minimize the risk of false-negative results. Effect-directed fractionation and analysis were applied to characterize and identify the toxicants that cause these effects. Major ethoxyresorufin-O-deethylase induction potency and mutagenicity were detected in different polyaromatic fractions, indicating different sets of toxicants that induce metabolic activation and mutagenicity. Dioxin-like halogenated aromatic hydrocarbons, including polychlorinated biphenyls, naphthalenes, dibenzo-p-dioxins and furans, and priority polycyclic aromatic hydrocarbons, contributed to Ah receptor-mediated activity only to a minor extent. Benzo[a]pyrene, benzo[a]fluoranthene, and perylene could be confirmed as important contributors to mutagenicity. The nonpriority pollutants 11H-indeno[2,1,7-cde]pyrene, a methylbenzo[e]pyrene, and a methylperylene were tentatively identified as major components, representing 82% of the peak area of a highly mutagenic fraction of the sediment extract. This suggests that hazard and risk assessment of complex environmental mixtures should make increasing attempts to identify and consider hazardous key pollutants rather than focusing on a priori-selected key pollutants alone.