The alpha2,6-sialylated blood group type 2H (ST2H) antigen (Fucalpha1-2(NeuAcalpha2-6)Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-Cer) is a fucoganglioside found in human colon cancer tissues. To elucidate an enzyme responsible for the ST2H antigen formation, we screened some partially purified candidate enzymes, alpha2,6-sialyltransferases, ST6Gal I and ST6Gal II, and alpha1,2-fucosyltransferases, FUT1 and FUT2 for their activities towards pyridylaminated type 2H (Fucalpha1-2Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-PA) or LS-tetrasaccharide c (LST-c: NeuAcalpha2-6Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-PA) as acceptor substrates. Here we show the ST6Gal I transfers NeuAc from the donor CMP-NeuAc to the terminal Gal of PA-type 2H, which formed the ST2H antigen, but the others could not synthesize it. Using a recombinant ST6Gal I, enzymatic reactions with two types of acceptors, PA-type 2H and PA-lacto-N-neotetraose (LNnT), were kinetically analysed. On the basis of catalytic efficiency (V(max)/K(m)), the specificity of ST6Gal I towards the PA-type 2H was estimated to be 42 times lower than that for PA-LNnT. The overexpression of ST6Gal I in human colon cancer DLD-1 cells effectively resulted in the ST2H antigen formation, as judged by LC-ESI-IT-MS. Many lines of evidence suggest the up-regulation of ST6Gal I in human colon cancer specimens. Collectively, these findings indicate that ST6Gal I is responsible for ST2H antigen biosynthesis in human colon cancer cells.