A Ca2+-dependent cysteine proteinase (calpain, EC 3.4.22.17) was found in the cystosolic fraction of bovine lens and purified to apparent homogeneity. The purified enzyme required 1 mM Ca2+ for its full activation and was composed of two subunits of Mr 80 000 and 29 000 as demonstrated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). This enzyme, when activated by Ca2+, degraded both A- and B-chains of alpha-crystallin, which were isolated also from bovine lens. SDS-gel electrophoresis of the digest revealed that the A-chain (Mr 19 500) was broken down to produce an 18-kDa polypeptide fragment and the B-chain (Mr 22 500) to produce a 19.5-kDa polypeptide fragment. No further cleavage occurred even upon prolonged incubation or after the second addition of the enzyme, indicating the uniquely limited proteolysis of each chain protein. The existence of calpastatin, an endogenous inhibitor protein specific for calpain, was also demonstrated in bovine lens cytosol.