Although piperonyl butoxide (PBO) is commonly used both in vivo and in vitro as an inhibitor of cytochrome P-450-dependent monooxygenase (MO) activity in a wide variety of species, the effect of PBO on the hepatic MO of fishes has never been characterized. The MO activity in hepatic microsomes from rainbow trout exposed to either 1 or 2 ppm PBO for 24 hr in a static system was induced to a similar level in both treatment groups. Conversely, when PBO was administered in a flow-through system to trout, the hepatic microsomes of treated animals contained MO activities that were induced in a dose-dependent manner. Furthermore, total cytochrome P-450 was significantly increased in the livers of trout treated in a flow-through system with 1 ppm or more of PBO. The in vitro inhibition kinetics of PBO toward the 7-ethoxycoumarin-O-deethylase (ECOD) and 7-ethoxyresorufin-O-deethylase (EROD) activities of hepatic microsomes from trout treated with beta-naphthoflavone (BNF) (100 mg/kg, ip) or PBO (4 ppm by flow-through) and untreated trout were compared with Dixon plots. With respect to ECOD activity, the slopes of Dixon plots from control, BNF- and PBO-treated animals were similar. However, the slopes of Dixon plots of EROD inhibition by PBO in microsomes from BNF- and PBO-treated trout were significantly different from each other. Treatment of trout with PBO in a flow-through system resulted in an increase in ECOD and EROD activity in hepatic microsomes while simultaneously decreasing their activity toward [14C]rotenone oxidation. These data suggest that the cytochrome P-450 isozyme composition in hepatic microsomes from PBO-treated rainbow trout may be qualitatively different from that of BNF-treated trout. Also, the activity of hepatic microsomes from PBO-treated trout toward a specific substrate may be either inhibited or induced.