Components of the hepatic mixed function oxidase (MFO) system are evaluated as a biomarker of contaminant exposure in fish from streams in Tennessee. The responses of several MFO components (ethoxyresorufin-O-deethylase (EROD) activity, cytochrome P-450 levels, and NADPH cytochrome c reductase activity) were comparable in fish from four pristine reference streams; EROD activity and cytochrome P-450 levels did vary over the year, with highest levels apparent in the autumn. Levels were lower in reproductively active females than in males during the same season. Fish from a stream impacted by an industrial effluent had EROD activities that were significantly higher than those from the reference stream; although the differences were greater in males during the warmer months. Reproductively active females from the contaminated stream also had higher EROD activity, but their enzyme activity was considerably lower than in males tested during the same season. Fish from the most severely impacted site did not have the highest EROD levels, possibly due to hepatotoxic damage to the liver; laboratory exposure of fish to an hepatotoxin reduced the capacity of the liver to respond to injection of benzo[a]pyrene, a known MFO inducer. MFO response in the fish was an informative biomarker of exposure, but the role of environmental, physiological, and toxicological factors on MFO response must be understood in order to properly interpret the biomarker response.