In this study, lavage fluid was fractionated from the superior joint space in patients with temporomandibular joint (TMJ) dysfunction. A hide powder azure protease assay was used to assess protease activity in lavage fluid. No correlation between a patient's pain and the level of protease activity was demonstrated. Latent as well as active proteases were detected in the sample lavage fluid. Latent matrix metalloproteinases (MMPs) were activated using trypsin. Stromelysin-1 was detected in an active form in lavage fluid by immunozymography. The presence of high molecular weight species with protease activity was also demonstrated. This study validates the presence of stromelysin-1 as well as other MMPs in TMJ lavage fluid and proposes a mechanism for their physiologic activation.