CTP-phosphoethanolamine cytidylyltransferase (ET) is the enzyme that catalyzes the formation of CDP-ethanolamine in the phosphatidylethanolamine biosynthetic pathway from ethanolamine. We constructed a Saccharomyces cerevisiae mutant of which the ECT1 gene, putatively encoding ET, was disrupted. This mutant showed a growth defect on ethanolamine-containing medium and a decrease of ET activity. A cDNA clone was isolated from a human glioblastoma cDNA expression library by complementation of the yeast mutant. Introduction of this cDNA into the yeast mutant clearly restored the formation of CDP-ethanolamine and phosphatidylethanolamine in cells. ET activity in transformants was higher than that in wild-type cells. The deduced protein sequence exhibited homology with the yeast, rat, and human CTP-phosphocholine cytidylyltransferases, as well as yeast ET. The cDNA gene product was expressed as a fusion with glutathione S-transferase in Escherichia coli and shown to have ET activity. These results clearly indicate that the cDNA obtained here encodes human ET.