We cloned a novel gene (aoxA) encoding amine oxidase (AOX) from Aspergillus oryzae. One cDNA clone showing extreme homology to the AOX-encoding genes was found in an expressed sequence tag (EST) library of A. oryzae. Molecular analysis revealed that the aoxA carried four exons interrupted by three introns and had an open reading frame encoding 672 amino acid residues. The deduced amino acid sequence showed about 83.5% identity to the Aspergillus niger AO-I. The strictly conserved residues for co-factor and copper binding in copper/quinine-containing, AOXs were also preserved at Tyr 405, His 456, His 458 and His 617 in the cDNA sequence. When the aoxA was overexpressed in the homologous hyperexpression system of A. oryzae, AOX activity in the transformant was enhanced 75-fold. An apparent molecular weight of 159.000 by gel filtration and a subunit molecular weight of 75,000 by SDS-PAGE of the purified enzyme were estimated, suggesting that the enzyme molecule is a homo-dimer similar to other copper/quinine containing AOXs. The A. oryzae AOXA preferentially oxidized aliphatic monoamines of C2-C6 rather than aromatic amines or diamines. From these results, the aoxA gene product obtained by homologous hyperexpression system of A. oryzae is undoubtedly a functional AOX.