The essential groups of three N-acetylglucosaminyltransferases (GnTs) of rat kidney were studied by using chemical modification and substrate protection methods. It was found that the amino and indolyl groups were the common essential groups of GnT-III, GnT-IV and GnT-V. Amino group was proposed to participate in the binding of common donor substrate--UDP-GlcNAc. Indolyl group might be the binding group of acceptor substrate--Gn(2)M(3)Gn(2)-PA heptosaccharide glycan for GnT-III and GnT-IV, and it might be the essential group but not substrate-binding for GnT-V. Guanidino group was also supposed to be one of the donor substrate-binding groups of GnT-III, but was a non-substrate-binding essential group for GnT-IV and a non-essential group for GnT-V, respectively. Carboxyl group might be the binding group of acceptor substrate for GnT-V, but the non-substrate-binding essential group for GnT-III and non-essential for GnT-V, respectively. In contrast, sulfhydryl, hydroxyl and imidazolyl groups were found to be non-essential groups of the three GnTs. The effects of amino sugars on GnT-III and GnT-V also showed the difference in essential groups in the active centers of GnT-III and GnT-V.