Thin-layer chromatography, reverse isotope dilution assay, and combined gas-liquid chromatography-mass spectrometry have demonstrated that an enzyme obtained from human lung catalyzes the in vitro methylation of N-methyltryptamine by S-adenosylmethionine to form N,N-dimethyltryptamine. Isolation of the product of the enzymic reaction and its identification were facilitated by use of carbon-14 and deuterium labeling.