A rapid, convenient assay to determine the activity of the flavin-containing monooxygenase is described. The method is based on direct analysis of quenched incubation mixtures by thin-layer chromatography and utilizes tritiated dimethylaniline as the substrate. The synthesis of the radiolabeled substrate is described. The usefulness of dimethylaniline N-oxide formation as a measure of flavin-containing monooxygenase activity was assessed using the purified hog liver enzyme, hog liver microsomes, and liver microsomes from untreated and phenobarbital-pretreated rats.