Based on the sequences of the highly conserved segments in the previously cloned sialyltransferases, a cDNA encoding a new type of Gal beta 1,3GalNAc alpha 2,3-sialyltransferase (ST3GalA.2) has been isolated from both mouse and rat brain cDNA libraries. The cDNA sequences included an open reading frame coding for 350 amino acids, and the primary structure of this enzyme suggested a putative domain structure consisting of four regions, like that in other glycosyltransferases. The deduced amino acid sequence of ST3GalA.2 (mouse) showed 76% identity in the active domain with that of the previously cloned mouse Gal beta 1,3GalNAc alpha 2,3-sialyltransferase (ST3GalA.1 (Lee, Y.-C., Kurosawa, N., Hamamoto, T., Nakaoka, T., and Tsuji, S. (1993) Eur. J. Biochem. 216, 377-385)). Northern blotting indicated that the expression of ST3GalA.2 mRNA is tissue-specific, it being prominent in brain and liver, while that in the other tissues is very low. This enzyme expressed in COS-7 cells exhibited transferase activity only toward the disaccharide moiety of Gal beta 1,3GalNAc of glycolipids as well as glycoproteins and oligosaccharides like ST3GalA.1, but showed a difference in acceptor substrate preference, i.e. asialo-GM1 and GM1 were much more suitable substrates for ST3GalA.2 than for ST3GalA.1.