The trypanosomatids are generally aberrant in their protein N-glycosylation pathways. However, protein N-glycosylation in the African trypanosome Trypanosoma brucei, etiological agent of human African sleeping sickness, is not well understood. Here, we describe the creation of a bloodstream-form T. brucei mutant that is deficient in the endoplasmic reticulum enzyme glucosidase II. Characterization of the variant surface glycoprotein, the main glycoprotein synthesized by the parasite with two N-glycosylation sites, revealed unexpected changes in the N-glycosylation of this molecule. Structural characterization by mass spectrometry, nuclear magnetic resonance spectroscopy, and chemical and enzymatic treatments revealed that one of the two glycosylation sites was occupied by conventional oligomannose structures, whereas the other accumulated unusual structures in the form of Glc alpha 1-3Man alpha 1 - 2Man alpha 1 - 2Man alpha 1 - 3(Man alpha 1 - 6) Man beta 1 - 4GlcNAc beta 1-4GlcNAc, Glc alpha 1 - 3Man alpha 1 - 2Man alpha 1 - 2Man alpha 1 - 3(GlcNAc beta 1-2Man alpha 1 - 6) Man beta 1 - 4GlcNAc beta 1 - 4GlcNAc, and Glc alpha 1 - 3Man alpha 1-2Man alpha 1 - 2Man alpha 1 - 3(Gal beta 1 - 4GlcNAc beta 1 - 2Man alpha 1 - 6) Man beta 1-4GlcNAc beta 1-4GlcNAc. The possibility that these structures might arise from Glc(1)Man(9)GlcNAc(2) by unusually rapid alpha-mannosidase processing was ruled out using a mixture of alpha-mannosidase inhibitors. The results suggest that bloodstream-form T. brucei can transfer both Man(9)GlcNAc(2) and Man(5)GlcNAc(2) to the variant surface glycoprotein in a site-specific manner and that, unlike organisms that transfer exclusively Glc(3)Man(9)GlcNAc(2), the T. brucei UDP-Glc: glycoprotein glucosyltransferase and glucosidase II enzymes can use Man(5)GlcNAc(2) and Glc(1)Man(5)GlcNAc(2), respectively, as their substrates. The ability to transfer Man(5)GlcNAc(2) structures to N-glycosylation sites destined to become Man(4-3)GlcNAc(2) or complex structures may have evolved as a mechanism to conserve dolichol-phosphate-mannose donors for glycosylphosphatidylinositol anchor biosynthesis and points to fundamental differences in the specificities of host and parasite glycosyltransferases that initiate the synthesis of complex N-glycans.