The cDNA encoding a new type of alpha 2,3-sialyltransferase (mST3Gal V) was cloned from mouse brain cDNA library by PCR-based cloning approach using a pair of degenerate primers deduced from the nucleotide sequence information of mouse ST3Gal III and IV. The predicted amino acid sequence of mST3Gal V showed 27.3% and 26.4% identity to mST3Gal III and IV, respectively. The recombinant soluble mST3Gal V fused with protein-A, which expressed in the culture media of COS-7 cells, showed activity toward lactosylceramide (LacCer), and synthesized GM3. The apparent Km value for LacCer was 9.3 microM. mST3Gal V did not exhibit any activity toward other substrates we tested in this study, including glycolipids, glycoproteins and disaccharides. The mST3Gal V cDNA transfected F28-7 cells, which express large amount of lactosylceramide and very small amount of GM3 at native stage, expressed a large amount of GM3. The ST3Gal V gene was strongly expressed in mouse brain and liver, which contained a large amount of ganglioside. The gene expression seemed to be coincident with ganglioside expression in mouse. Thus, we conclude that mST3Gal V is the fifth-type alpha 2,3-sialyltransferase carrying GM3 synthetic activity.