The enzyme CDP-diglyceride synthetase (CTP: phosphatidate cytidylyltransferase; EC 2.7.7.41) has been purified to 90% homogeneity from Escherichia coli cells that overproduce the enzyme 50-fold through the use of recombinant DNA technology. The purification required the use of different detergents at each step, illustrating the refractory hydrophobic nature of this protein. Apparent physical effects of EDTA on the enzyme were also utilized in the purification. The enzyme has an apparent minimum subunit mass of 27,000 daltons, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The amino acid composition of the protein was determined, and it correlates well with the theoretical protein product of the cds gene, the sequence of which is reported in the accompanying paper (Icho, T., Sparrow, C. P., and Raetz, C. R. H. (1985) J. Biol. Chem. 260, 12078-12083). The pure enzyme displays surface dilution kinetics when assayed in the presence of Triton X-100. As previously suggested on the basis of studies using partially purified preparations, the enzyme mechanism is sequential, and computer-calculated kinetic constants are reported herein. The substrate specificity of the enzyme is also investigated. This is the first time this enzyme has been purified to homogeneity from any source, despite the fact that it is essential for phospholipid biosynthesis in all organisms.