The cDNA encoding human NeuAc ${alpha}$2,3Gal$eta$ 1,3GalNAc GalNac ${alpha}$2,6-Sialyltransferase (hST6GalNac IV) was isolated by screening of human fetal liver cDNA library with a DNA probe generated from the cDNA sequence of mouse ST6Gal NAc IV (mkST6GalNAc IV). The cDNA sequence included an open reading frame coding for 302 amino acids, and comparative analysis of this cDNA with mST6GalNAc IV showed that each sequence of the predicted coding region contains 88% and 85% identifies in nucleotide and amino acid levels, respecively. The primary structure of this enzyme suggested a putative domain structure, like that in other glycosyltransferases, consisting of a short N-terminal cytoplamic domain, a transmembrane domain and a large C-terminal active domain. This enzyme expressed in COS-7 cells echibited transferase activity toward NeuAc ${alpha}$2,3Gal$eta$ 1,3GalNAc, fetuin and GM1b, although the activity toward the later is very low, no significant activity being detected toward Gal${eta}$ 1,3Gal NAc or asialofetuin, the other glycoprotein substrates tested. The $^{14}$ C-sialylated residue of fetuin sialylated by this enzyem with CMP-[$^{14}$C]NeuAc was sensitive to treatment with ${alpha}$2,8-specific sialidase of Vibrio cholerae but resistant to treatment with ${alpha}$2,3-specific sialidase (NaNase I), and ${alpha}$2,3- and ${alpha}$2,8-specific sialidase of Newcastle disease virus. These results clearly indicated that the expressed enzyme is a type of GalNAc ${alpha}$2,6-sialyltransferase like mST6GalNAc IV, which requires sialic acid residues linked to Gal${eta}$1,3GalNAc-residues for its activity.