The α-glucuronidase gene (aguA) of Aureobasidium pullulans NRRL Y-2311-1 was amplified by PCR and sequenced. Based on its deduced amino acid sequence, AguA was found to be a member of family 67 of the glycoside hydrolases. It shares greater than 60% identity and between 34% and 42% identity with fungal and with bacterial α-glucuronidases, respectively. The open reading frame lacks introns and encodes a polypeptide of 836 amino acids that contains a putative signal peptide of 15 amino acids resulting in a mature protein with a calculated molecular mass of 91.0kDa. A construct of the aguA gene encoding an additional C-terminal hexahistidine tag was cloned on an episomal plasmid under control of the ADH2 promoter and terminator and expressed in Saccharomyces cerevisiae Y294. The heterologous α-glucuronidase was purified to homogeneity by Ni-chelation affinity chromatography, and displayed an electrophoretic mobility of 157kDa on SDS–PAGE. Maximal activity was measured at 65°C and at pH 5 and pH 6. The enzyme had K m values in the millimolar range for the series of substrates from aldobiouronic acid to aldopentaouronic acid, but was unable to hydrolyze an internally substituted aldopentaouronic acid.