The purpose of these experiments was to examine the relationship between oxidation cataract and proteolysis in cultured rat lens. Hydrogen peroxide cataract showed insolubilization of protein, loss of 31 kDaβB1-crystallin polypeptide, decreases in soluble calpain, and increases in insoluble calpain. This suggested that calpain may be activated in hydrogen peroxide treated lenses, sinceβBl is a known calpain substrate, and calpain undergoes autolysis and degradation when activated. Furthermore, the cysteine protease inhibitor E64 was partially effective in preventing development of H2O2-cataract. E64 also prevented the loss of the 31 kDaβBl-crystallin polypeptide and decreased the loss of calpain in the lens. These results suggested that development of hydrogen peroxide induced cataract in rat lenses was associated with activation of calpain.