AbstractA periplasmic insulin‐cleaving proteinase (ICP)1), purified to its electrophoretic homogeneity in the SDS‐PAGE from the Gram‐negative bacteriumAcinetobacter calcoaceticus, was examined and compared in its properties with the protease III (protease Pi, pitrilysin, EC 3.4.99.44) ofEscherichia coliand the insulin‐destroying proteinase (IDE, insulinase, EC 3.4.99.45) from eucaryotes. The enzyme was proven to be a metalloprotease like protease III and IDE, as was shown by the inhibitory effects exerted by EDTA and o‐phenanthroline. Furthermore, dialysis against EDTA and o‐phenanthroline led to a complete loss of activity, which could be restored by addition of Co2+, and, to a lesser extent, but at a lower metal ion concentration by Zn2+Similar to protease III and IDE, ICP prefers the cleavage of small polypeptides (insulin, insulin B‐chain, glucagon) to the cleavage of proteins (casein, human serum albumin, globin) and was inactive against synthetic amino acid derivates (esters,p‐nitranilides, and furoylacroleyl substrates) of subtilisin, thermolysin, trypsin, and chymotrypsinThe peptide‐bond‐specificity of the ICP in the cleavage of the oxidized insulin B‐chain was investigated and the results were compared to the specificity of protease III ofE. coli, IDE, protease‐24,11, and thermolysin. Cleavage sites in the oxidized insulin B‐chain generated by ICP are Asn3‐Gln4, His10‐Leu11, Ala14‐Leu15, Leu17‐Vall8, Gly23‐Phe24, Phe24‐Phe25, and Phe25‐Tyr26. Principally, ICP cleaves between hydrophobic amino acids and amides. The ICP shares one of the only two cleavage sites with the p