UDP-N-Acetylglucosamine: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT-I) is an essential enzyme in the conversion of high mannose type oligosaccharide to the hybrid or complex type. The full length of the rat GnT-I gene was expressed in the filamentous fungus Aspergillus oryzae. A microsomal preparation from a recombinant fungus (strain NG) showed GnT-I activity that transferred N-acetylglucosamine residue to acceptor heptaose, Man(5)GlcNAc(2). The N-linked sugar chain of alpha-amylase secreted by the strain showed a peak of novel retention on high performance liquid chromatography that was same as a reaction product of in vitro GnT-1 assay. The peak of oligosaccharide disappeared on HPLC after beta-N-acetylglucosaminidase treatment. Mass analysis supported the presence of GlcNAcMan(5)GlcNAc(2) as a sugar chain of alpha-amylase from strain NG. Chimera of GnT-I with green fluorescent protein (GFP) showed a dotted pattern of fluorescence in the mycelia, suggesting localization at Golgi vesicles. We concluded that GnT-1 was functionally expressed in A. oryzae cells and that N-acetylglucosamine residue was transferred to N-glycan of alpha-amylase in vivo. A. oryzae is expected to be a potential host for the production of glycoprotein with a genetically altered sugar chain.