Keratoconus is a noninflammatory corneal disorder characterized by gradual stromal thinning and astigmatism. Altered degradation of corneal extracellular matrix is a suggested etiology for this disorder. In the present study we established keratocyte cultures from normal and keratoconus corneas and investigated the roles that matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMP, TIMP-2) may play. After chemical modification (reduction and alkylation) to remove the inhibitor and activation of enzyme with p-aminophenylmercuric acetate (APMA), keratoconus-conditioned media displayed a significant increase (p < 0.05) in the total potential gelatinolytic activity when compared with normal culture media treated in a similar manner. Basal levels of gelatinolytic activity in keratoconus culture media (no reduction, alkylation, or APMA treatment), determined by two different assay methods, tended to be about twice that of normal cell cultures. By zymography, both keratoconus and normal cultures showed identical enzyme patterns, which represented MMP-2 (72 kDa) in its proform and, depending on the treatment of the media, varying amounts of activated MMP-2 (65 kDa). This suggests that the increased gelatinolytic activity in keratoconus was not correlated with an increased appearance of either the 65-kDa-activated form of MMP-2 or a new MMP species. In addition, no differences in the amount of MMP-2 were detected that could account for the increased activities in keratoconus cultures. However, a relative decline in the detectable TIMP levels in keratoconus cultures resulted in an apparent three-fold increase in the ratio of MMP-2/TIMP. Northern blots showed no significant changes in mRNA levels for MMP-1, MMP-2, MMP-3, TIMP, or TIMP-2. These data suggest that a possible alteration in the interaction between MMP-2 and TIMP may play a role in the increased gelatinolytic activity seen in keratoconus tissues.