Calpain and its endogenous inhibitor, calpastatin, were isolated from erythrocytes of various mammals and their properties were compared. It has been widely believed that mammalian erythrocytes contain only mu-calpain. However, rat and human erythrocytes were found to contain two species of calpain, identified as mu-calpain and m-calpain from their elution positions on DEAE-cellulose column chromatography and their Ca(2+)-requirements. Thus, it is apparent that rat and human erythrocytes contain not only mu-calpain, but m-calpain as well. On the other hand, rabbit erythrocytes contain only mu-calpain. Western blot analysis showed that human and rabbit erythrocytes contain predominantly 70-kDa calpastatin (erythrocyte-type), but unnegligible amounts of 110-kDa calpastatin (tissue-type) are also present. Rat erythrocytes were shown to contain a calpastatin with a molecular mass of approx. 100 kDa almost exclusively; this molecular mass was in perfect coincidence with the mass of the calpastatin in rat lung. These results strongly suggest that rat erythrocytes contain a tissue-type calpastatin. No essential change in the calpain/calpastatin system during maturation of rabbit reticulocytes into mature erythrocytes was observed.