A colistin-inactivating enzyme, colistinase was produced by Bacillus polymyxa var. colistinus KOYAMA, a colistin-producing microorganism. The crude colistinase was fractionated as two components (colistinase I and II) by Sephadex G-50 gel filtration. Colistinase II was further purified and then, it showed as a single band in polyacrylamide disc gel electrophoresis. The molecular weight of colistinase II was about 20, 000 by Sephadex G-100 gel filtration and the isoelectric point was at about 8.3. Colistinase II cleaved specifically between the 2, 4-diaminobutyric acid of the side chain and 2, 4-diaminobutyric acid adjacent in the cyclic peptide portion of colistin molecule.