UDP-GlcNAc:Man alpha 1-3R beta 1-2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) catalyses the conversion of [Man alpha 1-6(Man alpha 1-3)Man alpha 1-6][Man alpha 1-3]Man beta-O-R to [Man alpha 1-6(Man alpha 1-3)Man alpha 1-6] [GlcNAc beta 1-2Man alpha 1-3]Man beta-O-R (R = 1-4GlcNAc beta 1-4GlcNAc- Asn-X) and thereby controls the conversion of oligomannose to complex and hybrid asparagine-linked glycans (N-glycans). GlcNAc-T I also catalyses the conversion of Man alpha 1-6(Man alpha 1-3)Man beta-O-octyl to Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta-O-octyl. We have therefore tested a series of synthetic analogues of Man"alpha 1-6(Man'alpha 1-3)Man beta-O-octyl as substrates and inhibitors for rat liver GlcNAc-T I. The 2"-deoxy and the 3"-, 4"- and 6"-O-methyl derivatives are all good substrates confirming previous observations that the hydroxyl groups of the Man"alpha 1-6 residue do not play major roles in the binding of substrate to enzyme. In contrasts, all four hydroxyl groups on the Man'alpha 1-3 residue are essential since the corresponding deoxy derivatives either do not bind (2'- and 3'-deoxy) or bind very poorly (4'- and 6'-deoxy) to the enzyme. The 2'- and 3'-O-methyl derivatives also do not bind to the enzyme. However, the 4'-O-methyl derivative is a substrate (KM = 2.6 mM) and the 6'-O-methyl compound is a competitive inhibitor (Ki = 0.76 mM). We have therefore synthesized various 4'- and 6'-O-alkyl derivatives, some with reactive groups attached to an O-pentyl spacer, and tested these compounds as reversible and irreversible inhibitors of GlcNAc-T I. The 6'-O-(5-iodoacetamido-pentyl) compound is a specific time dependent inhibitor of the enzyme. Four other 6'-O-alkyl compounds showed competitive inhibition while the remaining compounds showed little or no binding indicating that the electronic properties of the attached O-pentyl groups influence binding.