We cloned and characterized a novel gene (abfA) encoding alpha-L-arabinofuranosidase (alpha-L-AFase) from Aspergillus oryzae. One clone homologous to the alpha-L-AFase gene of Thermotoga maritima was found in an expressed sequence tag (EST) library of A. oryzae and a corresponding gene was isolated. Molecular analysis showed that the abfA gene carried six exons interrupted by five introns and had an open reading frame encoding 481 amino acid residues. The amino acid sequence similarity at active sites to the alpha-L-AFases from other organisms indicated that the alpha-L-AFase encoded by abfA was classified as a family 51 glycoside hydrolase. When the abfA was overexpressed in the homologous hyperexpression system of A. oryzae, a large amount of alpha-L-AFase was produced as intracellular protein. The apparent molecular mass of the purified enzyme was estimated to be 228,000 by gel filtration and that of its subunit as 55,000 by SDS-PAGE, suggesting that the enzyme is a tetramer. The enzyme hydrolyzed p-nitrophenyl-alpha-L-arabinofuranoside but not other p-nitrophenyl glycosides. These results demonstrated that the abfA gene encodes a functional alpha-L-AFase.