We valuated liver and skin tumor prevalence and biomarkers of exposure and response in brown bullhead (Ameiurus nebulosus) from three locations in the Anacostia River (Washington, DC, USA), a Chesapeake Bay region of concern. The Tuckahoe River (Maryland, USA) served as a reference. Each river was sampled in fall 2000 and spring 2001. In the Anacostia, prevalence of liver tumors was 50 to 68%, and prevalence of skin tumors was 13 to 23% in large (≥260 mm, age ≥3 years) bullheads. Liver and skin tumor prevalence was 10 to 17% and 0%, respectively, in small (150–225 mm, age 1–2 years) bullheads. Tuckahoe bullhead liver tumor prevalence was 0 to 3% (large) and 0% (small); none had skin tumors. Biliary polynuclear aromatic hydrocarbon (PAH)-like fluorescent metabolites and liver DNA adduct concentrations were elevated in large and small Anacostia bullheads. Mean adduct concentrations were 16 to 28 times higher than those in Tuckahoe fish. Chromatograms revealed a diagonal radioactive zone, indicating polycyclic aromatic compound (PAC)-DNA adducts. The biomarker data and the 10 to 17% liver tumor prevalence at ages 1 to 2 suggest that these year classes are likely to have a high prevalence as they reach age 3 and older. This study provides the strongest evidence to date of the role of PAHs in tumor development in Anacostia bullheads.