Using a purified protein and bisected acceptor oligosaccharides, we demonstrate that N-acetylglucosaminyltransferase (GnT)-V transfers a N-acetylglucosamine residue via a beta1,6-linkage to the bisected oligosaccharides. We also kinetically characterized the substrate specificity of GnT-V with respect to the bisected oligosaccharide. Although the K(m) values for the bisected acceptors were comparable to that for a non-bisected acceptor, the V(max) values for the bisected acceptors were much lower than that for the non-bisected acceptor. These findings suggest that the acceptor specificity of GnT-V is determined by the catalytic process rather than by its binding to the substrate. It was also found that the presence of the 2-N-acetyl group in the bisecting monosaccharide moiety plays a critical role in determining the catalytic efficiency of the enzyme.