yIntracellular environments are highly crowded due to the presence of various biomacromolecules. In this study, we estimated the property of the endoplasmic reticulum glucosidase II (G-II) under macromolecular crowding conditions. A crowded milieu that contains bovine serum albumin greatly enhanced the second trimming step (cleavage 2), which deglucosylates Glc(1)Man(9)GIcNAc(2), but not the first trimming step (cleavage 1), which removes the terminal glucose residue from GIC(2)Man(9)GIcNAC(2). A similar effect was obtained with ribonuclease A and high molecular weight polyethylene glycol 20 000. An analysis of CD spectra suggested that G-II enhanced its cleavage 2 activity through conformational change. We also investigated the effects of molecular crowding on other N-linked glycan-processing enzymes, UDP-Glc: glycoprotein glucosyltransfe rase and 1,2-alpha-mannosidase. Our results indicate that the kinetics of glycan processing under crowded conditions may be quite different from those measured in dilute buffers.