Using mammalian gene-overexpression system, in vitro catalytic activities of CMP-NeuAc:Gal beta 1-->4GlcNAc alpha 2-->6sialytransferase on glycosphinogolipid acceptors were analyzed. We transfected the mammalian expression vector containing the cDNA that was cloned from Daudi cells into COS-1 cells, and selected monoclonal transfectants in the presence of G418. Although the transfected alpha 2-->6sialytransferase can catalyze NeuAc transfer onto glycoprotein acceptors more than glycolipids based on kinetic analyses, the substantial synthesis of IV6NeuAc-nLcOse4Cer was observed and the activities were 7- to 9-times higher in the transfected cells than in the mock transfectants. In addition, the transfected COS-1 cells with alpha 2-->6sialytransferase cDNA were revealed to contain a higher amount of ganglioside that has the terminal NeuAc alpha 2-->6Gal sequence in the in situ situation than the mock transfectants. These results using transfectants, together with those using the purified enzyme protein, suggest that the alpha 2-->6sialytransferase enzyme from Daudi cells can also catalyze NeuAc transfer in alpha 2-->6 linkage onto glycosphingolipid acceptors.