A neutral extract from human leukocytes was shown to have proteolytic activity which could degrade triple-helical basement membrane collagen from bovine lens capsules into specific triple-helical sub-fragments. The enzymes responsible is not identical to the leukocyte collagenase active against interstitial collagen types I, II and III. After denaturation, the neutral protease reaction products showed three major peptides with molecular weights of 70,000, 50,000 and 30,000, by dodecylsulphate gel electrophoresis analysis. Electron microscopic observation and viscosity measurements showed the initial twice-pepsinized collagen to be comprised of intact rod-like molecules about 300 nm long. The fragments produced by the protease were also triple-helical and were resistant to the action of trypsin at 20 degrees C. The fragments were completely degraded by purified bacterial collagenase or by raising the reaction temperature above the melting temperature of the basement membrane collagen during incubation with leukocyte extract. Enzyme activity has its pH optimum between 7 and 9, EDTA, EGTA and 1,10-phenanthroline (metal-chelating agents) completely inhibited enzyme activity, as did serum. Partial inhibition of the activity was obtained with phenylmethylsulfonyl fluoride and soybean trypsin inhibitor.