To elucidate the relationship between the structure and function of CTP:phosphoethanolamine cytidylyltransferases (ECT, EC 2.7.7.14) in plants, we cloned and characterized the cDNA of anECT fromHordeum vulgare. ThisHvECT1 cDNA is 1783 bp long, and includes an open reading frame (ORF) of 1263 b that encodes a protein of 421 amino acids. The predicted protein sequence of HvECT1 is 73% identical to and 84% similar withArabidopsis thaliana AtECT1; it also shares 57, 55, and 37% similarities with human, rat, and yeast ECTs, respectively. Its 252-b 5′-noncoding leader contains a putative upstream ORF of 36 nucleotides, possibly encoding a putative peptide enriched in Ala and Pro residues. Alignment of the N-terminal and C-terminal halves of HvECT1 revealed a large internal repetitive sequence. Both halves contain the HXGH motif known to be involved in the catalytic activity of cytidylyltransferases. The RTXGVSTT sequence and Asp residues are also conserved. Our hydropathy profile showed that HvECT1 contains a signal sequence that is absent in yeast or animal ECTs. Results from reverse transcriptase-PCR indicated thatHvECT1 is expressed highly in the leaves, stems, and roots of one-week-old plants; its expression is not regulated by low temperatures. After transforming a yeast mutantect1 and labeling those transformants with radioactive ethanolamine, we identifiedHvECT1 cDNA throughin vivo analyses of the enzymatic reaction products.