It was reported before that cells of the trypanosomatid Leptomonas samueli incubated with [14C]glucose synthesized dolichol-P-P-linked Man9GlcNAc2 as the main and largest derivative. It is now reported that this protozoan is deficient in dolichol-P-Glc synthesis as judged from results obtained in a cell-free assay. We have structurally characterized several endo-beta-N-acetylglucosaminidase H sensitive oligosaccharides present in mature glycoproteins of this parasite. The compounds appeared to have the compositions Gal3Man9GlcNAc2, Gal2Man9GlcNAc2, Gal1Man9GlcNAc2, Man9GlcNAc2, Gal1Man8GlcNAc2, Man8GlcNAc2, Gal1Man7GlcNAc2, and Man7GlcNAc2. The galactose residues were in all cases in the furanose form and linked to mannoses in nonreducing ends. In the cases of Gal1Man8GlcNAc2 and Gal1Man7GlcNAc2, the galactose-substituted mannose units were the nonreducing residues originally present in the oligosaccharide transferred from dolichol-P-P (Man9GlcNAc2) and not the nonreducing termini generated by demannosylation of the latter oligosaccharide. Except for Gal3Man9GlcNAc2, the other galactosylated compounds appeared to be mixtures of several isomers.