The mu- and m-calpains are closely related Ca(2+)-dependent cysteine proteases having different in vitro Ca(2+) requirements ( K (d)), of approx. 25 and 325 microM respectively. The two isoforms are heterodimers of slightly different large (80 kDa) subunits and an identical small (28 kDa) subunit, so that the difference in K (d) values must reside in the large subunits. As assayed here, these K (d) values relate to the Ca(2+) required for the first phase of calpain activation and do not reflect the lower Ca(2+) then required by fully activated calpain. On the basis of sequence comparison and the X-ray structure of m-calpain, many m-type residues in the C-terminal EF-hand-containing domain IV were converted into the corresponding mu-type residues, but these mutations did not produce the expected decrease in K (d). In a series of hybrid (mu/m) large-subunit calpains, the K (d) values decreased progressively towards that of mu-calpain as the proportion of mu-type sequence increased from 0 to 90%. K (d) values cannot therefore be ascribed to one or a few specific intramolecular interactions, but reflect the global response of the whole molecule to Ca(2+) binding. Nonetheless, 25% of the difference in K (d) values between mu- and m-calpain can be ascribed to the N-terminal peptide of the large subunit, whereas the C-terminal EF-hand-containing domain IV accounts for 65% of the difference.