Reactions involving removal and addition of glucose to N-glycans in the ER (endoplasmic reticulum) are performed in higher eukaryotes by glucosidases I and II and the UDP-glucose:glycoprotein glucosyltransferase respectively. Monoglucosylated N-glycan structures have been implicated in glycoprotein folding or ER quality control. Components of the system appear across a range of organisms; however, the precise combination differs between organisms. We have identified putative components of the system in the protozoal organism Trypanosoma brucei by local alignment searching. The function of one of these components, a glucosidase II alpha-subunit homologue, has been confirmed by phenotyping a null mutant, and an ectopic expression cell line. A combination of MS, methylation linkage analysis, exoglycosidase digestion and partial acetolysis have been used to characterize three novel N-glycan structures on the variant surface glycoprotein of the null mutant. On the basis of our results, we propose that two N-glycan precursors are available for transfer to variant surface glycoprotein (variant 221) in the ER of T. brucei; only one of these precursors is glucosylated after transfer.