Rabbit uterine collagenase was purified from the medium of involuting uterus (1-2 days postpartum) in culture using ammonium sulfate fractionation, DEAE-cellulose, hepari n-af f inity, and high performance liquid chromatography. The enzyme was purified more than 1600 fold. Hybridoma cell - 1 ines produci ng monoclonal anti bodi es were prepared by fusing the spleen cells of mice immunized with the purified enzyme with mouse myeloma cells (Sp2/0-Ag14). The hybridoma cells were selected with HAT medium, cloned, and screened by ELISA. Antibody-producing ascites were prepared by injecting hybridoma cell-lines into the peritoneal cavities of mice. Western-blot analysis indicated that the antibodies recognized a polypeptide having a molecular weight of 52,000. The IgG isolated from the ascites inhibited the enzyme. Indirect immunofluorescent staining demonstrated that polymorphonuclear leukocytes (PMNs) in the superficial layer of alkali- burned corneas contained collagenase, whereas stronal cells and PMNs within the stroma were not stained by the antibodies. Our results suggest that collagenases produced by rabbit PMNs are different from those produced by fibroblasts from cornea. We hypothesize that PMNs in alkali-burned corneas secrete all or most of their collagenases by degranulation at the anterior surface of the cornea, and then continue to migrate into the deeper portion of the stroma.