Phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) catalyzes the conversion of norepinephrine to epinephrine, the last step of catecholamine biosynthesis. We have previously reported molecular cloning of cDNA encoding human PNMT and chromosomal localization of its gene (Kaneda et al., J. Biol. Chem., 263 (1988) 7672-7677). In this report, we isolated the chromosomal gene encoding mouse PNMT by cross-hybridization with the human PNMT cDNA. Mouse PNMT gene spanned about 1.8 kb and consisted of 3 exons. Primer extension analysis showed two putative transcription initiation sites. Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) revealed the expression of the mouse PNMT mRNA in brain (pons and medulla oblongata) and adrenal gland. Subsequently cDNA encoding mouse PNMT was amplified by RT-PCR and cloned into the plasmid vector. Mouse PMNT gene contained the protein-coding region of 885 bp (295 amino acids) with the predicted molecular weight of 32,627. The deduced amino acid sequence of mouse PNMT revealed the major difference in the N-terminal region, as compared to the human and bovine PNMT sequences. In the 5'-terminal region of the mouse PNMT gene, we found the existence of 23 bp direct repeat sequences, which was not observed in the corresponding regions of the human and bovine PNMT genes. The presence or absence of the direct repeats caused the major difference in the PNMT sequences among species. The typical TATA, GC, and CACCC boxes as well as several sequences homologous to glucocorticoids response elements (GRE) were located in the 5'-flanking region of the mouse PNMT gene.