The effect of acidic phospholipids on proteolysis of protein kinase C (PKC) by mu-calpain was examined at Ca++ concentrations ranging from 10(-7) to 10(-4) M. The gamma species, among the molecular species of PKC, was more susceptible to calpain than the alpha and beta (beta I/beta II) and was hydrolysed at Ca++ concentrations greater than or equal to 10(-6) M. Acidic phospholipids enhanced proteolysis of PKC gamma and lowered Ca++ concentrations required for it to the level below 10(-6) M. Among the phospholipids tested, phosphatidylinositol-bisphosphate showed the most prominent effect; phosphatidylinositol and phosphatidylserine were less effective. Polyphosphoinositides, hence, may constitute an essential structure in cell membranes for positive regulation of calpain activity.