Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide and structurally related compounds. There are large strain-dependent variations in the expression of NNMT activity in mouse liver during growth and development, raising the possibility of developmental regulation of the gene. Therefore, we set out to clone and structurally characterize the mouse NNMT gene, Nnmt. The gene spanned approximately 16 kb and consisted of three exons, 348 bp, 208 bp, and 487 bp in length, with an initial 1228-bp intron and a second intron that was approximately 14 kb in length. The locations of the splice junctions within the gene were highly conserved compared with those in genes for structurally related methyltransferase enzymes. The Nnmt gene contained no canonical TATA box sequences, but an "initiator" (Inr) sequence was located at the site of transcription initiation as determined by 5' rapid amplification of cDNAs ends. A promoter was located within the initial 750 bp of the 5' flanking region of the gene according to studies of the expression of a reporter gene in HepG2 cells. 5'-Flanking region sequences for mouse strains with high and low hepatic NNMT activity differed with regard to a series of nucleotide substitutions, insertions, and deletions, with the most striking difference being a 12-bp insertion/deletion. The Nnmt gene mapped to mouse chromosome 9 in an area of conserved synteny to human chromosome 11q, consistent with the localization of the human NNMT gene to 11q23. Cloning and structural characterization of the mouse Nnmt gene will make it possible to study molecular genetic mechanisms involved in the expression of this important methyltransferase.