Seven dog cytochromes p450 (p450s) were heterologously expressed in baculovirus-Sf21 insect cells. Of all enzymes examined, CYP1A1 exhibited high 7-ethoxyresorufin O-deethylase activity (low Km enzyme, 1 microM). CYP2B11 and CYP3A12 effectively catalyzed the N1-demethylation and C3-hydroxylation of diazepam (and its derivatives), whereas CYP3A12 and CYP2D15 catalyzed exclusively the N- and O-demethylation, respectively, of dextromethorphan. However, no saturation velocity curves for the N-demethylation of dextromethorphan (up to 500 microM) were achieved, suggesting a high Km for CYP3A12. In contrast to CYP3A12, the CYP2D15-dependent O-demethylation of dextromethorphan was a low Km process (Km = 0.7 microM), similar to that in dog liver microsomes (Km = 2.3 microM). CYP2D15 was also capable of metabolizing bufuralol (1'-hydroxylation), with a Km of 3.9 microM, consistent with that obtained with dog liver microsomes. CYP3A12 was shown to primarily oxidize testosterone at 16alpha-, 2alpha/2beta-, and 6beta-positions. Selectivity of CYP3A12 was observed toward testosterone 6beta-(Km = 83 microM) and 2alpha/2beta-hydroxylations (Km = 154 microM). However, the 16alpha-hydroxylation of testosterone was catalyzed by CYP2C21 also (Km = 6.4 microM for CYP2C21). Therefore, the 6beta- and 16alpha-hydroxylation of testosterone can potentially be employed as markers of CYP3A12 and CYP2C21 (at low concentration), respectively. CYP2C21 was also capable of catalyzing diclofenac 4'-hydroxylation, although some activity was detected with CYP2B11. Surprisingly, none of the p450s selectively metabolized (S)-mephenytoin 4'-hydroxylation. The results described herein are a first step toward the systematic evaluation of a panel of dog p450s and the development of dog p450 isoenzyme-selective marker substrates, as well as providing useful information on prediction and extrapolation of the results from in vitro to in vivo and from dog to human.