Our previous studies have demonstrated that chlorpromazine (CPZ) and imipramine (IMP) are metabolized appreciably via N-oxidation catalyzed by pulmonary microsomal flavin monooxygenase in the rat but not in the rabbit. The present work deals with the species differences in N-oxidase of pulmonary microsomes from male cat, dog, goat, guinea pig, hamster, mouse, pig, rabbit and rat. Although CPZ-N-oxidizing activities were generally higher than IMP-N-oxidizing activities, the CPZ-/IMP-oxidizing activity ratios were not constant among the species tested. Little or no activity was detected in the guinea pig and rabbit lung. The following ranking was assigned: mouse < hamster < pig < rat < dog < cat < guinea pig < goat < rabbit for CPZ, and pig < rat < mouse < dog < hamster < cat < guinea pig < goat < rabbit for IMP. Nitrosobenzene, a known inhibitor of N-oxidase reductase, inhibited rather than increasing the N-oxidation of both substrates. Therefore, it is unlikely that the marked species differences in pulmonary N-oxidase activities are due to differences in N-oxide reductase. Optimum pH for CPZ-N-oxidase was relatively broad over a range of 7.4–8.5 for rat and 8–9.5 for other species. The pH optima for IMP-N-oxidase ranged from 8.5 to 9.5. n-Octylamine accelerated CPZ-N-oxidation in most species. IMP-N-oxidation was affected to a lesser extent by this primary amine. DPEA (2,4-dichloro-6-phenyl-phenoxyethylamine) also stimulated the CPZ-N-oxidation in rat, pig and cat but inhibited IMP-N-oxidation in these species. These findings suggest that significant species differences exist in pulmonary N-oxidase activity and that the CPZ and IMP-N-oxidases may be represented by two different microsomal flavin amine oxidases.