As domestic animals such as cat, horse, and dog increasingly become the clinical targets for drug discovery programs, the need to understand how these animals metabolize xenobiotics becomes more important. In the present study, substrates and inhibitors that were reported to be selective for particular P450 isozymes were used as probes to study in vitro metabolism in horse, dog, cat, and human liver microsomes. Seven selective catalytic activity markers for cytochrome P450-mediated reactions were measured: phenacetin O-deethylase (P4501A1/2), coumarin 7-hydroxylase (P4502A6), tolbutamide hydroxylase (P4502C8/9), S-mephenytoin 4'-hydroxylase (P4502C19), dextromethorphan O-demethylase (P4502D6), chlorzoxazone 6-hydroxylase (P4502E1), and testosterone 6beta-hydroxylase (P4503A4). Metabolic activity was found in every species with each substrate. Under the conditions of this study, it was observed that no one species was more active for any given substrate. However, rather large interspecies differences were observed. There was no marked sex difference in the way the various species metabolized the different substrates. The effect of selective P450 inhibitors on the various activities was tested with furafylline (P4501A2), mouse monoclonal antibody inhibitory to CYP2A6, sulfaphenazole (P4502C9), tranylcypromine (P4502C19), quinidine (P4502D6), diethyldithiocarbamate (P4502E1), and troleandomycin (P4503A4). In most cases, these inhibitors were effective to varying degrees against the activity seen in horse, dog, and cat liver microsomes. However, even at high concentrations, furafylline did not inhibit phenacetin O-deethylase activity in cat and troleandomycin did not affect testosterone 6beta-hydroxylase activity in horse. Sulfaphenazole was not tested in dog and cat because of the low tolbutamide hydroxylase activity. Overall, these results show that there are also large interspecies differences in the way the selective P450 inhibitors affect the in vitro metabolism of the various substrates in horse, dog, and cat liver microsomes.