Incubation of Crithidia fasciculata cells with [U-14C] glucose led to the synthesis of Man-P-dolichol but not of Glc-P-dolichol. The main and largest dolichol-P-P-linked oligosaccharide formed was Man7GlcNAc2 whether labeling was performed in 5 mM sodium pyruvate or 5.5 mM glucose. The protein-linked, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides isolated from mature glycoproteins were Man7GlcNAc and Gal1Man6GlcNAc, the latter being a mixture of two isomers. All the galactose residues were present in the furanose configuration, as judged by their extreme lability to acid hydrolysis, by the products obtained upon mild periodate oxidation, and by their sensitivity to beta-galactofuranosidase. Labeling cells for short times or at low temperature yielded a protein-bound, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide whose composition was Glc1Man7GlcNAc, of transient existence, and that was mainly labeled in the glucose residue. The latter oligosaccharide was detected on paper chromatography only as a smearing of Man7GlcNAc and Gal1Man6GlcNAc when cells were labeled with [2-3H] mannose, thus indicating that it was only present in minute amounts. Protein-bound endo beta-N-acetylglucosaminidase H-resistant oligosaccharides liberated, upon a mild acid treatment, galactose residues and an unidentified substituent. The treatment rendered the oligosaccharides sensitive to endo beta-N-acetylglucosaminidase H, which liberated Man7GlcNAc and two isomers of Man6GlcNAc. An almost similar mechanism of protein N-glycosylation, including the existence of galactofuranose residues in N-linked oligosaccharides, was found to occur in Crithidia harmosa.