The primary structure of a novel phosphate-containing oligosaccharide, isolated from T. reesei cellobiohydrolase I, was determined by NMR techniques. The new compound has the same structure as GlcMan7GlcNAc2, but it is extended by one alpha-mannopyranosyl unit (Man-P) through a phosphate link. Three different heteronuclear (31P-1H) NMR techniques were used to prove that the phosphate links the glycosidic site of Man-P with C-6 of unit Man-B. The presence of mannoses linked through a phosphate diester resembles glycosyl synthesis in yeast.