Collagenase (EC 3.4.24.3) is released from bovine gingival explants in vitro as a zymogen. The zymogen does not hydrolyze collagen and does not form a complex with alpha2-macroglobulin (alpha2-M). It elutes in gel filtration with an apparent molecular weight of approx. 80 000. 2. Incubation of the zymogen with trypsin results in a 15 000-20 000 dalton decrease in molecular weight and imparts to the enzyme the ability to hydrolyze collagen and to form a complex with alppha2-M. 3. The zymogen can be completely separated from the active enzyme to alpha2-M. Likewise, the zymogen can be harvested from cultures supplemented with serum.