The ability of aquatic organisms to efficiently biotransform and eliminate xenobiotics is an important strategy for reducing body burdens and thereby avoiding toxic effects of xenobiotic exposure. We have developed a plexiglass chamber that isolates the different xenobiotic elimination routes in fish, enabling the collection of biotransformation products over time from the gills, skin, urine, and feces of rainbow trout (Oncorhynchus mykiss). Trout were exposed to [14C]-labeled pentachlorophenol (PCP) in the physiological chamber using two experimental treatments: (1) intraperitoneal (i.p.) injection of PCP into fish having no chemical or effluent preexposure (controls) and (2) i.p. injection of PCP into fish following preexposure to a complex pulp mill effluent for 2 weeks. Elimination of PCP residues was monitored from all routes for 24 to 48 h. Specific PCP biotransformation products were identified and quantified in urine, feces, and bile using high performance liquid chromatography (HPLC) to determine the effects of pulp mill effluent preexposure on the routes and rates of PCP elimination in exposed trout. This study demonstrates the utility of using the physiological chamber for studying the mechanisms of xenobiotic biotransformation and elimination.