The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide-specific phospholipase C (PLC) is absolutely dependent on Ca2+. The PH domain truncated catalytic core of rat phospholipase C delta1 (PLC-delta1) has Ca2+ binding sites in its catalytic and C2 domains, and potential Ca2+ binding sites in two EF-hands. A catalytically inactive PLC-delta1 catalytic core bound with low affinity to PIP2-containing vesicles in the presence of Ca2+. A mutant PLC-delta1 has been engineered which lacks the C2 domain Ca2+ binding site and the surrounding loops known as the jaws. Isothermal calorimetric titration showed four Ca2+ ions bind to the wild-type PLC-delta1 catalytic core in solution but only one binds to the C2 domain jaws deletion mutant. The activity and Ca2+ dependence of wild-type and mutant phospholipase Cs were determined using substrate incorporated in detergent micelles and in large unilamellar vesicles. The activities of wild-type and mutant were identical to each other in both assay systems. Wild-type and the C2 jaws deletion mutant of PLC have Hill coefficients of 1.12-1.16 with respect to [Ca2+]. We conclude that a single Ca2+ bound to the catalytic domain is entirely responsible for the Ca2+ dependence of the basal activity of PLC-delta1.