Cardiac Na+,Ca2+ exchange is activated by a mechanism that requires hydrolysis of adenosine triphosphate (ATP) but is not mediated by protein kinases. In giant cardiac membrane patches, ATP acted to generate phosphatidylinositol-4,5-bisphosphate (PIP2) from phosphatidylinositol (PI). The action of ATP was abolished by a PI-specific phospholipase C (PLC) and recovered after addition of exogenous PI; it was reversed by a PIP2-specific PLC; and it was mimicked by exogenous PIP2. High concentrations of free Ca2+ (5 to 20 mu M) accelerated reversal oi the ATP effect, and PLC activity in myocyte membranes was activated with a similar Ca2+ dependence, Aluminum reversed the ATP effect by binding with high affinity to PIP2. ATP-inhibited potassium channels (K-ATP) were also sensitive to PIP2, whereas Na+,K+ pumps and Na+ channels were not. Thus, PIP2 may be an important regulator oi both ion transporters and channels.