The pullulanase (EC 126.96.36.199) of Bacillus acidopullulyticus was purified using anion exchange chromatography and preparative isoelectric focusing. The purified preparation migrated as a single protein band upon SDS gel electrophoresis and its molecular weight was estimated to be 102,000. Two main activity bands having pl‐values 5.0 and 5.2 were detected by analytical isoelectric focusing. The enzyme was purified 4‐fold with the yield 38% by this two‐step purification procedure. The purified pullulanase showed maximal activity at 50°C and pH 5 and was slightly activated by Ca2+. It was stable at 50°C but totally lost its activity at 60ºC in one hour. This purified pullulanase efficiently hydrolyzed the α‐1,6‐glucosidic bonds of pullulan and gelatinized starch.