The effect of a calcium-binding protein (CaBP) isolated from rat liver cytosol on pnos-phorylase α activity in the hepatic particulate glycogen was investigated. The particulate glycogen phosphorylase α activity was significantly increased by addition of Ca²⁺ in the range of 1.0-10³ μM. This increase was not prevented by the presence of N- (6-aminohexyl) -5-chloro-1-napthalenesulfonamide (W-7 100 μM), an inhibitor of calmodulin. The elevation of phosphorylase α activity by 10 μM Ca²⁺ addition was significantly inhibited by the presence of CaBP at a concentration of greater than 3.0 μM. This inhibition was complete at 7.0 μM CaBP. CaBP itself had no effect on the enzyme activity. Of various metals (20 μM) used, addition of Zn²⁺ caused a significant decrease of the particulate glycogen phosphorylase α activity, while Mg²⁺ Mn²⁺ and Cd²⁺ had no effect on the enzyme activity. The presence of 3.5 μM CaBP did not block the inhibition of the enzyme activity by Zn²⁺ addition, indicating that CaBP uniquely reverses the Ca²⁺ effect. The present study suggests that CaBP regulates the effect of Ca²⁺ on phosphorylase α activity in the hepatic particulate glycogen of rats.