Penicillin amidase of Escherichia coli was purified abot 3,200 folds by ammounim sulfate fractionation, DEAE-cellulose, CM-cellulose, and Sephadex G-100 colum chromatography. The overall recovery yield was 57%. As compared with the previous purification method, the purification factor and the recovery yield increased about 56 and 1.4 folds, respectively. The purified enzyme was found to be homogeneous in polyacrplamide gel electrophoresis. The molecular weight determined by SDS-gel electrophoresis was 55,000. The specific activity of penicillin amidase for benzylpenicillin was 2.22 ${mu}$moles/sec/mg protein and the catalytic rate constant was 122 $sec^{-1}$. The optimum pH and temperature were 8.5 and $50^{circ}C$, respectively. This enzyme was rapidly deactivated at temperature above $50^{circ}C$ and pH above 9.0. Almost all of the metal ions did not affect the enzyme activity except for $Hg^{+2}$ ion. The Michaelis constant for benzylpenicillin was 2.9 mM, the substrate inhibition constants for 6-APA and phenylacetic acid were 10 mM and 4.1 mM, respectively A steady state equation based on the four-step ordered reaction with an acyl intermediate was derived. The inhibition modes of both products based on this equation were discussed with respect to the present experimental results.