Pulmonary coumarin 7-hydroxylase, testosterone hydroxylase and other P450-mediated activities were compared in the mouse and rat. Coumarin 7-hydroxylase activity was 20 pmol/mg/min. in mouse and 4 pmol/mg/min. in rat lung microsomes. Liver values were 180 (mouse) and 1 (rat) pmol/mg/min. Km values of rat and mouse lung coumarin 7-hydroxylase were about 1 microM whereas the rat liver Km value was > 100 microM. Phenobarbital and 3-methylcholanthrene did not affect rat lung (or liver) coumarin 7-hydroxylase activity. Anti-Cyp2a-5 antibody effectively inhibited mouse and rat lung coumarin 7-hydroxylase and testosterone 15 alpha-hydroxylations but failed to block these activities in the rat liver. In immunoblot analysis anti-Cyp2a-5 antibody recognized the 50-kDa Cyp2a-4/5 protein in mouse lung microsomes. A P450 protein co-migrating with Cyp2a-5 was also detected in rat lung microsomes. Cyp2a-5 cDNA probe hybridized with a 1.8-kb mRNA species in rat lung RNA fraction. The hybridization signal was not increased by 3-methylcholanthrene or phenobarbital. These data suggest that the mouse lung expresses Cyp2a-5 which differs from the liver enzyme only in its regulation and that the rat lung contains a P450 isoform(s) belonging to the 2A subfamily which may be orthologous with the mouse Cyp2a-4/5 catalyzing coumarin 7-hydroxylase and testosterone 15 alpha-hydroxylase activities. The recently reported rat lung CYP2A3 (Kimura et al.) gene product is a candidate for the observed coumarin 7-hydroxylase activity in the rat lung.