Biotransformation plays a key role in detoxification, bioactivation and bioaccumulation of xenobiotics in fish. Biotransformation capabilities in fish, however, are not as thoroughly characterized as in mammals. In this study, basal level activities of 7-ethoxyresorufin O-dealkylase (EROD), 7-methoxyresorufin O-dealkylase (MROD), 7-pentoxyresorufin O-dealkylase, chlorzoxazone 6-hydroxylase, testosterone 6beta-hydroxylase, lauric acid 11-hydroxylase, and glutathione S-transferase in freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss) and rat were obtained and compared. All the activities, when normalized to cellular protein concentrations, were significantly lower in rainbow trout hepatocytes than those in rat hepatocytes. Rainbow trout cytochrome P450 (CYP) 2B-, 2E1-, and 3A-like activities were respectively 14-, 18.4-, and 11.8-fold lower than rat. The smallest difference between the two species was CYP1A-type activities (EROD and MROD, 3.4- and 3.7-fold lower in rainbow trout, respectively). Our results suggest that the relative importance of CYP1A enzymes (among CYP subfamilies) in the biotransformation of xenobiotics in rainbow trout is likely much greater than that in mammals.