A new, high molecular weight (66,000 daltons) inhibitor of collagenase (LCI) has been isolated and partially characterized. It accounted for 20% of the collagenase-inhibitory activity in the supernatants of rabbit chondrocytes cultured in 10% acid-treated fetal bovine serum (ATFBS). LCI was stable to 60 degrees C and sensitive to reduction and alkylation. Unlike a low molecular weight collagenase inhibitor, similar to Tissue Inhibitor of Metallo-Proteinases (TIMP), it did not bind to concanavalin A-Sepharose 4B.