To study the mechanisms of activation of human neutrophil gelatinase, the enzyme has been purified using a combination of chromatography on a DEAE-Sephacel and a gelatin-peptide-Sepharose column. On reducing SDS-polyacrylamide-gel electrophoresis the purified gelatinase ran as a single band of about 94,000 Da, and had a specific activity of 5624.4 units/mg of enzyme protein. When latent gelatinase was treated with trypsin, cathepsin G, neutrophil elastase, HgCl2 or urea, its activity was enhanced and the enzyme was processed and converted into species of the lower molecular mass. Upon activation, the protein band of 94,000 Da of reduced latent gelatinase underwent a decrease of about 6,000-12,000 Da. Formation of the species of lower molecular mass during urea activation could be blocked by the addition of EDTA.