A Mr 92,000 metalloprotease, originally observed in neutrophils, has been found to be secreted by various normal and malignant cells of fibroblastic, hematopoietic, and epithelial origin. The responsiveness of the various cell types to the tumor promoter phorbol ester (phorbol myristate acetate) to secrete this enzyme and a corresponding Mr 72,000 gelatinase has been determined using gelatin zymograms. The latent zymogen form of the Mr 92,000 enzyme has been purified from phorbol myristate acetate-stimulated HT1080 human fibrosarcoma cells using sequential gelatin-Sepharose affinity chromatography and gel filtration. Selective elution from gelatin-Sepharose allows for a distinct separation of the Mr 92,000 gelatinase from the Mr 72,000 gelatinase. A fraction of the tumor cell derived latent Mr 92,000 enzyme is isolated as an apparent complex with human tissue inhibitor of metalloproteases, which is partially dissociated in sodium dodecyl sulfate and completely dissociated upon reduction of disulfide bonds and upon p-aminophenylmercuric acetate treatment. Organomercurial treatment rapidly allows for autoactivation of the proenzyme to active Mr 83,000 and Mr 75,000 species. At physiological pH, the enzyme rapidly degrades gelatin into small fragments and slowly cleaves native type V collagen at an apparent single site. Native type IV collagen is degraded to a much lesser extent. The NH2-terminal amino acid sequence of the Mr 92,000 proenzyme has been determined and is distinct from the Mr 72,000 gelatinase/type IV collagenase which is constitutively produced by fibroblasts. The Mr 92,000 enzyme is also immunologically distinct from the Mr 72,000 enzyme but immunologically cross-reactive with the neutrophil, high molecular weight gelatinase. The Mr 92,000 enzyme constitutes a distinct member of the matrix metalloprotease family. Its substrate specificity implies a broad physiological role, acting on basement membrane type V collagen as well as on denatured (gelatinized) collagens and thus may be involved in the invasive and migratory phenotype of human cells.