Collagenolytic enzyme activity has been demonstrated in middle ear effusions from patients suffering from otitis media with effusion. Collagenase activity was characterized using different biochemical techniques. Various chemical and physical parameters were titrated for optimal enzyme activity. Bivalent cations activated the enzyme with Ca2+ as the most potent activator. Chelators such as EDTA reduced the enzyme at low concentrations. The enzyme was found to have a higher specific activity in mucoid effusions than in serous and had characteristics similar to granulocyte derived collagenase from human leukocytes. Possible relationships between the presence of collagenase activity and tissue destruction in the middle ear after otitis media with effusion are discussed. The collagenase activity showed wide ranges within different categories of middle ear effusions.