CTP:phosphocholine cytidylyltransferase (CCT) is a key regulatory enzyme in phosphatidylcholine (PtdCho) biosynthesis, and in mammals, there are two distinct genes that encode enzymes that catalyze this reaction. This work defines the structures of both the murine CCT genes (Pcyt1a and Pcyt1b) and identifies a new CCT protein, CCTbeta3, with a unique amino terminus that arises from an alternate initiation exon. CCTalpha is expressed in all tissues, and is most abundant in liver, kidney and heart. A second CCTalpha transcript is described that initiates from a separate untranslated exon that is most highly expressed in testis. The CCTbeta isoforms are most highly expressed in brain and reproductive tissues. CCTbeta3 is not expressed in embryonic brain tissues, but is a significant transcript in the adult. These data suggest unique roles for the CCT protein isoforms in the differential regulation of PtdCho biosynthesis in specific tissues.