We have found previously that L-type Ca2+ channel run-down in cell-free patches is partially (10-28%) reversed by calpastatin (CS) and have suggested that CS, an endogenous inhibitor of calpain, has a Ca2+-channel-regulating function. CS is composed of repetitive domains 1-4 (calpain-inhibitory domain) and domain L (a domain whose function is unknown). We therefore investigated which domain of CS was involved in the regulation of Ca2+ channel activity in guinea pig cardiac myocytes using the patch-clamp technique. After the patches were excised into inside-out mode in basic internal solution, the Ca2+ channel activity ran down to 0.45% of the control level recorded in the cell-attached mode. Application of human recombinant full-length CS (25 microM) and domain L (25 microM) restored the Ca2+ channel activity to 13 and 19% of the control level, respectively, while the channel activity was not restored by CS domain 1 (25 microM) (0.66%). Mouse CS domain XLL (25 microM), a complex of domain XL and domain L, restored the calcium channel activity to 11% of the control level. These results suggested that the Ca2+ channel-regulating function of CS is located in domain L. This study is the first description of the function of CS domain L.