CTP:phosphocholine cytidylyltransferase (CCT) is a key regulator of phosphatidylcholine biosynthesis, and only a single isoform of this enzyme, CCTalpha, is known. We identified and sequenced a human cDNA that encoded a distinct CCT isoform, called CCTbeta, that is derived from a gene different from that encoding CCTalpha. CCTbeta transcripts were detected in human adult and fetal tissues, and very high transcript levels were found in placenta and testis. CCTbeta and CCTalpha proteins share highly related, but not identical, catalytic domains followed by three amphipathic helical repeats. Like CCTalpha, CCTbeta required the presence of lipid regulators for maximum catalytic activity. The amino terminus of CCTbeta bears no resemblance to the amino terminus of CCTalpha, and CCTbeta protein was localized to the cytoplasm as detected by indirect immunofluorescent microscopy. Whereas CCTalpha activity is regulated by reversible phosphorylation, CCTbeta lacks most of the corresponding carboxyl-terminal domain and contained only 3 potential phosphorylation sites of the 16 identified in CCTalpha. Transfection of COS-7 cells with a CCTbeta expression construct led to the overexpression of CCT activity, the accumulation of cellular CDP-choline, and enhanced radiolabeling of phosphatidylcholine. CCTbeta protein was posttranslationally modified in COS-7 cells, resulting in slower migration during polyacrylamide gel electrophoresis. Expression of CCTbeta/CCTalpha chimeric proteins showed that the amino-terminal portion of CCTbeta was required for posttranslational modification. These data demonstrate that a second, distinct CCT enzyme is expressed in human tissues and provides another mechanism by which cells regulate phosphatidylcholine production.