A thermostable cyclodextrinase (EC 3.2.1.54) from Bacillus stearothermophilus HY-1 was purified to homogeneity by disc-electrophoresis after sonication disruption, ammonium sulfate fractionation, DEAE-cellulose(DE32) column chromatography, hydroxyapatite chromatography, Sephadex G150 gel-filtration, and a-cyclodextrin-AH-Sepharose 4B affinity chromatography. The enzyme was purified 230-fold with 21.2% of activity recovery. The optimal substrates of the enzyme were alpha-, beta-, and gamma-cyclodextrins and linear maltooligosaccharides, and the final product was mainly maltose. The enzyme could hydrolyze pullulan to produce panose, It could also hydrolyze soluble starch, amylose, and amylopectin, but not glycogen. The Km and Vmax for alpha-, beta-, and gamma-cyclodextrins were 1.79, 1.67, and 2.50 mg/mL, and 336, 185, and 208 mu mol/mg/min, respectively. The molecular weight of the enzyme was 61,000 by SDS-gel-electrophoresis. The isoelectric point was pH 5.0. The enzyme was most active at pH 6.2 and 55 degrees C, and it was strongly inhibited by CU2+, Hg2+, Zn2+, Pb2+, and slightly by Fe3+. The effect of some protein modification reagents on the activity of the enzyme suggested that tryptophan and histidine residue(s) may be located at the active site, The amino acid composition of the enzyme was also determined.