AbstractA carcinogenicity‐ and mutagenicity‐directed fractionation approach was used to identify the carcinogenic compounds in contaminated sediments that are putatively responsible for the high prevalence of tumors in bottom‐dwelling fish from Hamilton Harbour, Ontario. Mutagenic activity was detected with Ames tester strains (TA98, TA100) in relatively nonpolar fractions of sediment extract containing PAHs and nitrogen‐containing aromatic compounds (NCACs). These fractions were also carcinogenic in an in vivo carcinogenicity bioassay with rainbow trout (Oncorhynchus mykiss). When a more polar extract fraction was tested for mutagenicity and carcinogenicity, weak mutagenic activity was detected with anO‐acetyltransferase‐enriched Ames tester strain (YG1024), and weak carcinogenic activity was detected in the rainbow trout assay. These data indicate that PAHs in contaminated Hamilton Harbour sediments are potent fish carcinogens, but it is also evident that other organic compounds in the sediment, such as NCACs and nitroarenes, may contribute to carc