CTP:phosphocholine cytidylyltransferase (CCT) catalyzes a rate-controlling step in the biosynthesis of phosphatidylcholine (PtdCho). Multiple CCT isoforms, CCTα, CCTβ2, and CCTβ3, are encoded by two genes, Pcyt1a and Pcyt1b. The importance of CCTα in mice was investigated by deleting exons 5 and 6 in the Pcyt1a gene using the Cre-lox system. Pcyt1a^−/− zygotes failed to form blastocysts, did not develop past embryonic day 3.5 (E3.5), and failed to implant. In situ hybridization in E11.5 embryos showed that Pcyt1a is expressed ubiquitously, with the highest level in fetal liver, and CCTα transcripts are significantly more abundant than transcripts encoding CCTβ or phosphatidylethanolamine (PtdEtn) N-methyl transferase, two other enzymes capable of producing PtdCho. Reduction of the CCTα transcripts in heterozygous E11.5 embryos was accompanied by upregulation of CCTβ and PtdEtn N-methyltransferase transcripts. In contrast, enzymatic and real-time PCR data revealed that CCTβ (Pcyt1b) expression is not upregulated to compensate for the reduction in CCTα expression in adult liver and other tissues from Pcyt1a^{+/ −} heterozygous mice. PtdCho biosynthesis measured by choline incorporation into isolated hepatocytes was not compromised in the Pcyt1a^{+/ −} mice. Liver PtdCho mass was the same in Pcyt1a^+/+ and Pcyt1a^+/− adult animals, but lung PtdCho mass decreased in the heterozygous mice. These data show that CCTα expression is required for early embryonic development, but that a 50% reduction in enzyme activity has little detectable impact on the operation of the CDP-choline metabolic pathway in adult tissues.