Calpain requires Ca2+ for both proteolysis of its substrates and interaction with its endogenous inhibitor, calpastatin. The mechanism of inhibition of calpain by calpastatin has remained unsolved, although Nishimura and Goll [J. Biol. Chem. 266, 11842-11850 (1991)] reported that autolyzed calpain fragments containing calmodulin-like domains (CaMLDs) bound to an immobilized calpastatin column. We investigated the correlation between CaMLD-binding and calpain inhibition using immobilized columns of gene-engineered CaMLDs derived from the human mu-calpain large subunit and various recombinant calpastatin mutants. Among the four internally repetitive inhibitory domains of calpastatin, each having conserved regions A, B, and C, only domains 1 and 4 showed the binding activity. The region B deletion mutant of domain 1, retaining the CaMLD-binding ability, no longer had the calpain inhibition activity, and became susceptible to proteolysis. In contrast, a synthetic oligopeptide of region B with moderate calpain inhibition activity did not bind to the column. Domain 3 acquired the binding ability on substitution of region A with that of domain 1. These results suggest that calpain inhibition and binding to the CaMLDs are not correlated or mediated by different subdomains of calpastatin.