Our laboratories have previously demonstrated that normal rabbit periarticular ligaments, anterior cruciate ligaments (ACL), medial collateral ligaments (MCL) and patellar tendon (PT) secrete collagenase. In this current study we examined these connective tissues following an immobilization period of 4 weeks. In the ligaments producing collagenase, activity was expressed only in the control, not in the immobilized joint. Control and experimental patellar tendon samples produce collagenolytic activity, suggesting that the expression of enzyme is less affected in tendons as compared to ligaments. Characterization of these collagenases was carried out using an antiserum directed against rabbit synovial collagenase. We demonstrated that ligament (ACL) and tendon (PT) collagenases cross react with this antibody in a double immunodiffusion assay. Protein blots of PT, ACL and MCL collagenases identified one major species (Mr = 45,000) and a minor species (Mr = 50,000) of immunoreactive proteins in all three connective tissues. Differences between control and experimental enzyme levels appear to be due to less collagenase protein being produced by immobilized ligaments.