Two cDNA clones encoding NeuAc␣2,3Gal1,3GalNAc GalNAc␣2,6-sialyltransferase have been isolated from mouse brain cDNA libraries. One of the cDNA clones is a homologue of previously reported rat ST6GalNAc III according to the amino acid sequence identity (94.4%) and the substrate specificity of the expressed recombinant enzyme, while the other cDNA clone includes an open reading frame coding for 302 amino acids. The deduced amino acid sequence is not identical to those of other cloned mouse sialyltransferases, although it shows the highest sequence similarity with mouse ST6GalNAc III (43.0%). The expressed soluble recombinant enzyme exhibited activity toward NeuAc␣2, 3Gal1,3GalNAc, fetuin, and GM1b, while no significant activity was detected toward Gal1,3GalNAc or asialofetuin, or the other glycoprotein substrates tested. The sialidase sensitivity of the 14 C-sialylated residue of fetuin, which was sialylated by this enzyme with CMP-[ 14 C]NeuAc, was the same as that of ST6GalNAc III. These results indicate that the expressed enzyme is a new type of GalNAc␣2,6-sialyltransferase, which requires sialic acid residues linked to Gal1,3GalNAc residues for its activity; therefore, we designated it mouse ST6GalNAc IV. Although the substrate specificity of this enzyme is similar to that of ST6GalNAc III, ST6GalNAc IV prefers O-glycans to glycolipids. Glycolipids, however, are better substrates for ST6GalNAc III.Sialic acids are key determinants of carbohydrate structures that play important roles in a variety of biological functions, like cell-cell communication, cell-substrate interaction, adhesion, and protein targeting. The transfer of sialic acids from CMP-Sia 1 to the terminal positions of the carbohydrate groups of glycoproteins and glycolipids is catalyzed by a sialyltransferase. Although roles of sialic acids have been proposed in the regulation of many biological phenomena, the purpose of this structural diversity remains largely obscure. To determine the meaning of the diversity of and the regulatory mechanism for the sialylation of glycoconjugates, it is necessary to obtain information on the enzymes themselves and the gene structure of sialyltransferases. Each sialyltransferase exhibits strict specificity for acceptor substrates and linkages (3-6). Although three linkages, Sia␣2,6Gal, Sia␣2,3Gal, and Sia␣2,6GalNAc, are commonly found in glycoproteins (7), and two, Sia␣2,3Gal and Sia␣2,8Sia, occur frequently in gangliosides (8), each of these linkages has been found in both gangliosides and glycoproteins (8 -10).So far, the cloning of three members of the ␣2,6-sialyltransferase family (ST6GalNAc I, II and III) has been reported (11-14). The cDNAs of ST6GalNAc I and II were cloned from both chick (11, 12) and mouse (13,62).2 The overall amino acid sequence identity of chick ST6GalNAc I is 30.5% to chick ST6GalNAc II, 43.2% to mouse ST6GalNAc I, and 33.6% to mouse ST6GalNAc II, and that of mouse ST6GalNAc I is 29.6% to mouse ST6GalNAc II and 28.3% to chick ST6GalNAc II, and that of chick ST6GalNAc II is 57.3% ...