Calpastatin molecule contains four repeated inhibition domains, each having highly conserved internal regions A, B and C. The synthetic oligopeptides of regions A and C had no calpain inhibition activity while region B oligopeptide showed weak inhibition activity. Real-time biomolecular interaction analysis using a BIAcore instrument revealed that the bacterially expressed calmodulin-like domain of the calpain large subunit (L-CaMLD) and that of the small subunit (S-CaMLD) interacted, in a Ca(2+)-dependent fashion, preferentially with the immobilized synthetic oligopeptide of region A and that of region C, respectively. Calmodulin showed no specific binding to these oligopeptides. The tripartite structure of the calpastatin functional domain may confer the specific interactions with the protease domain and the two CaMLDs of calpain.