A cDNA of human Gal beta 1,3GalNAc alpha 2,3-sialytransferase (hST3Gal II) which has been known to exhibit much more acceptor substrate preference for glycolipid than for O-linked oligosaccharides of glycoproteins, was isolated from the human liver cDNA library by plaque hybridization using the cDNA of mouse ST3Gal II (mST3Gal II) cloned previously as a probe. Comparative analysis of this cDNA with mST3Gal II indicates 89 and 94% homologies in the nucleotide and amino acid levels, respectively, between the two sequences in the predicted coding region. Northern analysis indicated that the expression of hST3Gal II mRNA is tissue-specific, it being prominent in skeletal muscle and heart, while that in lung and kidney is very low. This enzyme expressed in COS cells showed a similar activity with that of mST3Gal II.