Thielavia terrestris is a soil-borne thermophilic fungus whose molecular/cellular biology is poorly understood. Only a few genes have been cloned from the Thielavia genus. We detected an extracellular glucoamylase in culture filtrates of T. terrestris and cloned the corresponding glaA gene. The coding region contains five introns. Based on the amino acid sequence, the glucoamylase was 65% identical to Neurospora crassa glucoamylase. Sequence comparisons suggested that the enzyme belongs to the glycosyl hydrolase family 15. The T. terrestris glaA gene was expressed in Aspergillus oryzae under the control of an A. oryzae alpha-amylase promoter and an Aspergillus niger glucoamylase terminator. The 75-kDa recombinant glucoamylase showed a specific activity of 2.8 mumol/(min.mg) with maltose as substrate. With maltotriose as a substrate, the enzyme had an optimum pH of 4.0 and an optimum temperature of 60degreesC. The enzyme was stable at 60degreesC for 30 min. The K-m and k(cat) of the enzyme for maltotriose were determined at various pHs and temperatures. At 20degreesC and pH 4.0, the enzyme had a K-m of 0.33 +/- 0.07 mM and a k(cat) of (5.5 +/- 0.5) X 10(3) min(-1) for maltotriose. The temperature dependence of k(cat)/K-m indicated an activation free energy of 2.8 kJ/mol across the range of 20-70degreesC. Overall, the enzyme derived from the thermophilic fungus exhibited properties comparable with that of its homolog derived from mesophilic fungi.