We have proposed that cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylation is carried out by a multienzyme system involving CMP-NeuAc hydroxylase (the terminal enzyme of the system), cytochrome b5, and an NADH-dependent cytochrome b5-reducing factor (Kozutsumi, Y., Kawano, T., Yamakawa, T., and Suzuki, A. (1990) J. Biochem. 108, 704-706). The CMP-NeuAc hydroxylase was purified to homogeneity from the cytosolic fraction of mouse liver, using ion exchange columns, a Red-Sepharose column, and a soluble cytochrome b5-immobilized Sepharose column. The purified enzyme exhibited a single band (64 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a single peak (58 kDa) on gel permeation chromatography, indicating that it is composed of a single polypeptide chain. The absorption spectrum did not indicate the presence of a heme prosthetic group in the enzyme. Atomic absorption spectrometry and an inhibition test using an iron chelator indicate that the enzyme contains non-heme iron as an electron acceptor. A reconstitution experiment with the purified CMP-NeuAc hydroxylase, soluble cytochrome b5, and recombinant NADH-cytochrome b5 reductase revealed that these three factors are essential for the reaction. The hydroxylase exhibited high affinity to CMP-NeuAc (Km = 5 microM) and was greatly stabilized by CMP-NeuAc. The molecular activity of the enzyme (approximately 500/min) is much lower than that reported for NADH-cytochrome b5 reductase, suggesting that the activity or amount of hydroxylase is rate-limiting in CMP-N-glycolylneuraminic acid (NeuGc) biosynthesis. These results, together with the previous observation that the level of CMP-NeuAc hydroxylase activity was associated with the expression of NeuGc in various tissues, support the notion that the enzyme is the key for regulation of the overall velocity of CMP-NeuAc hydroxylation and consequently for the expression of NeuGc-containing glycoconjugates.