N-Benzyl-4-substituted anilines are metabolized by N-debenzylation, aniline-ring hydroxylation and N-oxidation. Factors affecting metabolism of these secondary anilines have been studied in vitro. Enzymes responsible for C- and N-oxidation are present mainly in the liver, but also occur in lung and kidney and are localized in the endoplasmic reticulum. Quantitative and qualitative species differences were observed in microsomal N-benzylaniline metabolism. In hamsters and mice N-debenzylation is the major route of metabolism. In guinea pig and rabbit, ring hydroxylation is the major pathway whereas the rat uses both pathways to equal extents. The hamster has particularly high N-oxidase activity. A sex difference in microsomal N-benzylaniline metabolism is evident in rats but not mice. Kinetic constants for C- and N-oxidation of N-benzylanilines are reported.