A cDNA encoding a new alpha 2,8-sialyltransferase (ST8Sia III), which exhibits activity toward the Sia alpha 2,3Gal beta 1, 4GlcNAc sequences of N-linked oligosaccharides, was cloned from mouse brain by means of the polymerase chain reaction-based approach. The predicted amino acid sequence of ST8Sia III showed 27.6 and 34.4% identity with those of so far cloned mouse alpha 2,8-sialyltransferases, i.e. GD3 synthase (ST8Sia I) and STX (ST8Sia II), respectively. Transfection of the protein A-fused ST8Sia III gene into COS-7 cells led to alpha 2,8-sialyltransferase activity toward sialylated glycoproteins and alpha 2,3-sialylated glycosphingolipids, such as alpha 2,3-sialylparagloboside and GM3. However, the kinetic properties of ST8Sia III revealed that it is much more specific to N-linked oligosaccharides of glycoproteins than glycosphingolipids. The expression pattern of the ST8Sia III gene was clearly different from those of other alpha 2,8-sialyltransferase genes. The expression of the ST8Sia III gene was tissue and stage specific. The ST8Sia III gene was expressed only in brain and testis, and it appeared first in 20 postcoitum embryonal brain and then decreased. Therefore, the new alpha 2,8-sialyltransferase is closely involved in brain development.