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Two isozymes of calpain, mu-calpain and m-calpain, were purified from rabbit, monkey, human and rat tissues to homogeneity and the apparent molecular masses of the large and small subunits of each calpain species were compared directly. While the molecular masses of the small subunits were the same (28 kDa), those of the large subunits were different depending on the calpain type and animal species: Rabbit mu (79 kDa), rabbit m (75 kDa), monkey mu (79 kDa), monkey m (74 kDa), human mu (78 kDa), human m (73 kDa), rat mu (75 kDa), and rat m (74 kDa). Ca2+-sensitivity of monkey mu-calpain was lower than that of rabbit mu-calpain, but m-calpains from rabbit and monkey shared a similar Ca2+-dependency. Immunoreactivities of rabbit, monkey and rat m-calpains towards anti-rabbit m-calpain monoclonal antibodies were different depending on the antibody species, showing the existence of common and different antigenic sites in these three m-calpains. While monkey mu-calpain still showed weak cross-reactivity with anti-rabbit mu-calpain monoclonal antibodies, rat mu-calpain failed to react with any antibody examined, except for the monoclonal 1D10A7 which reacted with mu- and m-calpains from any animal species. Peptides generated by V8 protease digestion were similar between mu-calpains or m-calpains from rabbit and monkey but, again, the reactivity of monkey calpain peptidesto anti-rabbit calpain antibodies was weak, especially to those of mu-calpain.