Analyses of biomarkers in fish were used to evaluate exposures among locations and across time. Two types of references were used for comparison, an upstream reference sample remote from known point sources and regional exposure criteria derived from a baseline of fish from reference sites throughout Ohio, USA. Liver, bile, and blood were sampled from white suckers (Catostomus commersoni) and common carp (Cyprinus carpio) collected during 1993 and 1996 in the Ottawa River near Lima, Ohio. Levels of exposure were measured for petroleum by naphthalene-type metabolites, combustion by-products by benzo[a]pyrene-type metabolites, coplanar organic compounds by ethoxyresorufin-O-deethylase (EROD) activity, and urea by blood urea nitrogen (BUN) levels. The four biomarkers analyzed proved effective in determining differences between reference and polluted sampling sites, between geographically close (<0.5 km) sites, and between sampling years at sites common in both years. Calculated exposure criteria levels of the polycyclic aromatic hydrocarbon bile metabolites were found to be a conservative approximation of levels from a designated reference site and could thereby permit comparison of biomarker levels of fish from the Ottawa River to a regional reference level. Polycyclic aromatic hydrocarbon bile metabolite and EROD activity levels were more reflective of spatial patterns of contamination than BUN, although all biomarkers indicated differences over time. Biomarkers from white suckers seemed to be more responsive in detecting changes in contaminant levels than the same biomarkers from common carp. Lower levels in 1996 of all biomarkers at many sites suggested lower exposures than in 1993 and could be indicative of some improvement over the period.