A genomic DNA segment and cDNAs encoding an extracellular exoinulinase from Aspergillus niger strain 12 were cloned and sequenced. Southern blot analysis indicated that the exoinulinase gene (inuE) was present as a single copy in the genome. An open reading frame of 1611 bp was interrupted by a single intron of 60 bp, and encoded a 19-amino acid signal peptide and a 518-amino acid mature protein. The mature protein contained a single Cys residue and nine potential N-linked glycosylation sites. Three distinct transcription start points were observed at positions -41 (A), -35 (A), and -31 (A) from the start codon. The 5'-noncoding region had a putative TATA box at position -75 (TATAAA). Transcription of the inuE gene was induced by inulin or sucrose and repressed by fructose or glucose. The inuE cDNA was functionally expressed under the control of the alcohol oxidase gene promoter in the methylotrophic yeast Pichia pastoris. The deduced amino acid sequence of the inuE gene product was 91% identical to that of an exoinulinase from Aspergillus awamori. A neighbor-joining tree showed that exo- and endoinulinases found in Aspergillus and Penicillium spp. have independently evolved the respective hydrolytic activities toward terminal and internal beta-2,1-fructofuranosidic linkages in inulin.