In the present study, experimental control of the formation of bisecting GlcNAc was investigated, and the competition between -1,4-GalT (UDP-galactose:N-acetylglucosamine -1,4-galactosyltransferase) and GnT-III (UDP-N-acetylglucosamine:-D-mannoside -1,4-N-acetylglucosaminyltransferase) was examined. We isolated a -1,4-GalT-I single knockout human B cell clone producing monoclonal IgM and several transfectant clones that overexpressed -1,4-GalT-I or GnT-III. In the -1,4-GalT-Isingle knockout cells, the extent of bisecting GlcNAc addition to the sugar chains of IgM was increased, where -1,4-GalT activity was reduced to about half that in the parental cells, and GnT-III activity was unaltered. In the -1,4-GalT-I transfectants, the extent of bisecting GlcNAc addition was reduced although GnT-III activity was not altered significantly. In the GnT-III transfectants, the extent of bisecting GlcNAc addition increased along with the increase in levels of GnT-III activity. The extent of bisecting GlcNAc addition to the sugar chains of IgM was significantly correlated with the level of intracellular -1,4-GalT activity relative to that of GnT-III. These results were interpreted as indicating that -1,4-GalT competes with GnT-III for substrate in the cells.