Cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylase, which is the key enzyme for the synthesis of N-glycolylneuraminic acid (NeuGc), has been purified from the cytosolic fraction of mouse liver, as described in our previous paper. The amino acid sequences of the purified CMP-NeuAc hydroxylase, and peptides obtained by lysylendopeptidase digestion, were used to synthesize specific oligonucleotide primers. A mouse cDNA clone of the enzyme was obtained by a combination of the polymerase chain reaction and rapid amplification of cDNA ends. The sequence of the clone contained an open reading frame coding for a protein of 577 amino acids with a predicted molecular mass of 66 kDa. The deduced sequence included the amino acid sequences obtained for the purified enzyme and peptides, and a complete match was obtained for 159 residues. The enzyme has neither a signal peptide sequence nor a membrane spanning domain, which is consistent with localization of the enzyme in the cytosol. Transfection of a cDNA construct to COS-1 cells increased the enzyme activity and the amount of NeuGc. Comparison of the sequence with GenBank data indicated that no similar sequence has been reported so far. Northern blot analysis of various mouse tissues with the enzyme cDNA as a probe indicated that expression of NeuGc is related to the level of CMP-NeuAc hydroxylase mRNA. On Southern blot analysis with the same probe, cross-hybridizing bands were detected in the human and fish genomes.