Indolethylamine N-methyltransferase (INMT) catalyzes the N-methylation of tryptamine and structurally related compounds. This reaction has been studied because of its possible role in the in vivo synthesis of psychoactive compounds or neurotoxins and has been characterized biochemically in preparations of rabbit lung. Therefore, we set out to purify rabbit lung INMT, to clone and express its cDNA, and to clone and structurally characterize its gene as steps toward understanding the function and regulation of this enzyme. Rabbit lung INMT was purified and partial amino acid sequence was obtained. A polymerase chain reaction-based approach was then used to clone a rabbit lung INMT cDNA with a 792-base pair open reading frame that encoded a 263-amino acid protein with a predicted molecular mass of 29 kDa. When the cDNA was expressed in COS-1 cells, the encoded protein catalyzed the methylation of tryptamine and structurally related compounds, and was inhibited by two products of the reaction, S-adenosyl-L-homocysteine (AdoHcy) and N,N-dimethyltryptamine, as well as antimigraine drugs that are structurally related to N,N-dimethyltryptamine. Northern blot analysis demonstrated the presence of 2.0-kilobase mRNA species in rabbit lung, liver and, at lower levels, in brain. The cDNA was then used to clone the rabbit INMT gene. That gene had three exons and was structurally similar to the genes for nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase in several species. Cloning and expression of a rabbit lung INMT cDNA and cloning of the rabbit INMT gene represent important steps toward determination of the function and regulation of this mammalian methyltransferase enzyme.