Corneal buttons were obtained from eight young adult patients with advanced keratoconus and were incubated in an organ culture system. The medium was assayed for collagenolytic activity using 14C-labeled reconstituted native collagen fibers as substrate. The assay was performed with and without prior collagenase activation with trypsin. The results indicated that the medium obtained from keratoconic corneas showed higher levels of collagenolytic activity compared with normal control corneas. We suggest that the level of collagenase activation may be a pathogenic factor in the development of keratoconus.