The aims of the present study were to determine cytochrome P450 enzyme activity in six strains of experimental rodents (n = 5/sex/species): ICR, C57BL/6 and DBA/2 mice; Sprague Dawley and Wistar rats; and Dunkin Hartley guinea pigs. After animals were treated with the typical inducers β-naphthoflavone (BNF), dexamethasone (DEX) and phenobarbital (PB), the levels of O-dealkylation of ethoxyresorufin (EROD), methoxyresorufin (MROD), pentoxyresorufin (PROD) and benzyloxyresorufin (BROD) activity were determined using responsive catalytic reactions to study CYP1A1, CYP1A2 and CYP2B, respectively. A maximal induction of EROD and MROD was found in BNF-treated animals from all strains (2.4- to 15.1-fold) except DBA/2 (0.9- to 1.8-fold). C57BL/6 mice had the strongest BNF-induced EROD (15.1-fold) and MROD (8.3-fold) activities. No differences in BNF-induced EROD and MROD activities were observed between males and females. However, the EROD activity of Wistar rats and the MROD activity of Sprague Dawley rats were higher in males than females. DEX induced PROD activity only in mice (1.3- to 7.1-fold), but not in rats and guinea pigs (0.2- to 1.1-fold). However, induction of BROD activity was found in DEX-treated mice and rats (1.5 to 12.5-fold), but not in guinea pigs (0.3 to 0.4-fold). PB caused a significant elevation of PROD (1.7- to 10.4-fold) and BROD (31- to 13.2-fold) activities in all the animals. PB-induced BROD activity was higher in females than males in Sprague Dawley rats. These observations strongly suggest that the choice of experimental animal strain, species and inducer is of critical importance for studies of drug metabolism and interaction. Copyright © 2012 John Wiley & Sons, Ltd.