To investigate the potential of filamentous fungi to synthesize N-glycans that are convertible to a mammalian type, in vitro glycosylation assays were performed. Recombinant human N-acetylglucosaminyltransferase I, human beta-1,4-galactosyltransferase and rat alpha-2,6-sialyltransferase were successively used to mimic part of the mammalian glycosylation synthesis pathway. High-mannose carbohydrates on Trichoderma reesei cellobiohydrolase I were converted to a hybrid mammalian-type structure. Successful modification varied markedly with the strain of T. reesei used to produce cellobiohydrolase I. In vitro pretreatment of fungal glycoproteins with Aspergillus saitoi alpha-1,2-mannosidase improved subsequent hybrid formation. It was, however, not possible to trim all fungal oligosaccharides to an acceptor substrate for mammalian glycosyltransferases. With T. reesei RUTC 30, capping glucose residues and phosphate groups were shown to be responsible for this lack of trimming. N-glycan processing in T. reesei apparently involves different steps, including alpha-1,2-mannosidase trimmings, and thus resembles the first mammalian glycosylation processes. The alpha-1,2-mannosidase trimming steps can be exploited for further in vitro and/or in vivo synthesis of complex oligosaccharides on (heterologous) glycoproteins from filamentous fungi.