N-Glycoloylneuraminic acid (Neu5Gc) is synthesized as its CMP-giycoside by the action of CMPN-acetylneuramlnic acid (CMP-Neu5Ac) hydroxylase. This enzyme is a soluble cytochrome bs-dependent monooxygenase and has been purified to apparent homogeneity from pig submandibular glands by precipitation with N-cetyN,N,N-trimethylam-monium bromide and fractionation on Q-Sepharose, Cibacron Blue 3GA-Agarose, Reactive Brown 10-Agarose, Hexyl-Agarose and Superose S.12. This procedure resulted in an 8960-fold purification of the hydroxylase with a recovery of 0.8%. The molecular mass of this protein was shown to be 65 kDa on SDS-PAGE and 60 kDa as determined by gel filtration on Superose S.12, which suggests that the enzyme is a monomer. The purified CMP-Neu5Ac hydroxylase is activated by FeSO4 and inhibited by iron-binding reagents such as o-phenanthroline, KCN, Tiron and ferro-zine. An apparent Km of 11 μM was determined for the substrate CMP-Neu5Ac using purified hydroxylase in the presence of Triton X-100-solubilized microsomes. In a reconstituted system consisting of purified hydroxylase, cytochrome b5, cytochrome b5 reductase and catalase, an apparent Km of 3 μM was measured. The apparent Kmfor cytochrome b5 in this system was 0.24 μM. Immunization of a rabbit with enriched and purified hydroxylase led to an antiserum that inhibited CMP-Neu5Ac hydroxylase activity and reacted with the purified 65 kDa protein on a Western blot after SDS-PAGE. Antibodies specific for this 65 kDa protein were isolated and showed a strong reaction with the purified CMP-Neu5Ac hydroxylase from mouse liver after immunoblotting. Initial experiments with this monospecific antibody suggest that the activity of the hydroxylase in a particular tissue correlates with the amount of immuno-reactive protein.