To study the regulation of collagen degradation in periodontium, human gingival homogenate was incubated at 36 degrees C and the release of hydroxyproline was assayed as a measure of collagenase activity. Phenylmethylsulfonyl fluoride, soybean trypsin inhibitor and serum albumin inhibited the in vitro collagenolysis while p-aminophenylmercuric acetate, a sulfhydryl reagent, increased the degradation. When latent collagenase obtained from gingival fibroblast culture was added to the incubation a marked increase in the collagen degradation was found. This increase was prevented by phenylmethylsulfonyl fluoride. The data suggests that collagenase may exist in gingiva partly in a latent form and its activation may be brought about by 2 mechanisms. A serine proteinase present in tissue may activate collagenase by producing a limited cleavage, or the activation may occur through a reaction that involves the sulfhydryl groups of the collagenase molecule.