Microsomes prepared from equine submandibular glands and incubated with tritium-labelled AcCoA incorporated acid-insoluble radioactivity in a manner dependent on time, protein, membrane integrity and AcCoA concentration, with incorporation being optimal at 37°C and pH 6.6. Under the experimental conditions used a K M of 32.1 μM for AcCoA and a Vmax of 1.2 pmol/mg protein x min was obtained. The incorporation of acid-insoluble radioactivity was also inhibited by CoA in a competitive manner (K i=240 μM), as well as by para-chloromercuribenzoate, 3′-dephospho-CoA, 5′-IDP, 5′-ADP, ß-NAD and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate. We demonstrate here that this incorporation of radioactivity into endogenous sialic acid is due to the action of an AcCoA:sialate-4-O-acetyltransferase [EC 2.3.1.44]. Radio thin-layer chromatography analyses of propionic acid-released sialic acids showed that the incorporation of radioactivity correlated with the formation of a radiolabelled species that co-migrated with authentic Neu4,5Ac2. Saponification experiments using NaOH, mouse hepatitis virus strain S and Influenza C/JJ/50 virus also showed that the transfer of [3H]acetyl groups from [3H]AcCoA to endogenous sialic acid acceptors was occurring exclusively at carbon 4 of the pyranose ring.