We isolated human ST6GalNAc III cDNA clones. The typical cDNA clones predicted a type II membrane protein of 305 amino acids with a short cytoplasmic transmembrane domain of sixteen amino acids and a catalytic domain of 280 amino acids. A short form clone predicted a protein of 240 amino acids lacking 65 amino acids including the transmembrane portion. The alternative usage of the second exon seemed to generate these two transcripts. Both had two common regions found among sialyltransferases cloned so far, i.e. sialyl motif L and sialyl motif S. Alignments of human, mouse and rat orthologs indicated that high homologies, i.e. 85-95% identity among these species at amino acid levels. We analyzed the expression pattern and substrate specificity of the product, demonstrating a very restricted expression pattern and a high substrate specificity. Northern blotting revealed that hST6GalNAc III is expressed in kidney and brain as a single band at 3.2 kb. In enzyme assay of the long form, the transfer of sialic acid onto alpha2,3-sialylated acceptor substrates, i.e. GM1b and sialyl lactotetraosylceramide, was observed. hST6GalNAc III also showed sialyltransferase activity toward O-glycans (but not N-glycans) in fetuin.