Recent laboratory studies with nontarget fish species have shown that the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) exhibits estrogenic activity through binding to rainbow trout (Oncorhynchus mykiss) hepatic estrogen receptors and induction of vitellogenin in hepatocyte cultures. In addition, mixed function oxygenase (MFO) activity associated with exposure to field formulations has been attributed in part to the presence of chloro-nitro-trifluoromethyl-dibenzo-p-dioxin impurities. To investigate the environmental effects associated with these findings, the temporal and spatial patterns of MFO activity and vitellogenin induction were monitored in three nontarget fish species following a TFM field treatment. Elevated MFO activity was detected as early as 1 day in caged rainbow trout and activity in trout, wild white sucker (Catostomus commersoni), and longnose dace (Rhinichthys cataractae) peaked 2 or 3 days after treatment. Highest activities were observed in fish exposed closest to lampricide application points and declined with increasing distance downstream. After 18 days, MFO activity was reduced but remained almost sevenfold reference values at several sites. Plasma vitellogenin was not detected in caged trout sampled 6 and 18 days post-treatment, and dioxin impurities were not detected in sediments after treatment. It was concluded that laboratory testing underestimated the duration of MFO activity under field conditions and that an assessment of formulation exposure during sensitive life stages represents an area for further study.