A synthetic 27-residue peptide corresponding to exon 1B of the endogenous inhibitor calpastatin contains a well-conserved region and has an ability to inhibit the cysteine endopeptidase calpain specifically. We examined the solution structure of this peptide in DMSO-d6 by two-dimensional 1H NMR spectroscopy. Although regular secondary structures such as alpha-helix and beta-sheet were not found, the region from Ile18 to Arg23 formed a well-defined structure with a type I beta-turn. This region coincided well with the highly conserved region of calpastatin. The result strongly suggests that this turn structure is essential for the inhibitory activity of calpastatin.