Penicillin acylase from B. megaterium was purified by the steps including ammonium sulfate fractionation, Sephadex G-100 gel filtration, hydroxyapatite absorption and DEAE-cellulose chromatography. The results showed that the specific activity of the purified enzyme was 25U/mg protein with 58% activity recovery. Only one protein band was detected by 5%-20% gradient PAGE or SDS-PAGE, it indicated that the penicillin acylase was purified to homogeneity and no subunit was found. The molecular weight of the enzyme was about 140kD. The kinetic parameters determined with NIPAB as substrate were Km= 6. 2 ×1 04M and Vm = 1 . 2 × 10￣(-4)M.The purified enzyme was much stable. The optium pH and temperature was 9. 0 and 47 ℃ respectively. The divalent metal ions such as Mn￣(2+), Zn￣(2+), Cd￣(2+), Ni￣(2+) and chelating agent EDTA showed no obvious effect on the enzyme activity.