Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes the conversion of phosphatidylethanolamine to phosphatidylcholine in the mammalian liver via three sequential methylations. In the present studies, we cloned and characterized the murine gene for PEMT2, the isoform of the enzyme that localized to the mitochondria-associated membrane. The structure of the gene was determined by analysis of two lambda and three P1 genomic clones, and compared to the known rat PEMT2 cDNA sequence. Southern blotting of mouse genomic DNA indicated that PEMT2 is a single-copy gene. The gene spans at least 35 kb, with seven exons and six introns. Two transcription start sites, 139 and 148 base pairs upstream of the translation start site, were detected by primer extension and reverse transcriptase-polymerase chain reaction. These experiments indicated that the PEMT2 gene is transcribed from a single promoter. Finally, the PEMT2 gene was localized to mouse chromosome 11 by interspecific backcrossing. These experiments represent the first cloning and characterization of a full-length mammalian gene involved in phospholipid biosynthesis.