Cardiac Na/Ca exchange (NCX1) inactivates in excised membrane patches when cytoplasmic Ca is removed or cytoplasmic Na is increased. Exogenous phosphatidylinositol-4,5-bis-phosphate (PIP2) can ablate both inactivation mechanisms, while it has no effect on inward exchange current in the absence of cytoplasmic Na. To probe PIP2 effects in intact cells, we manipulated PIP2 metabolism by several means. First, we used cell lines with M1 (muscarinic) receptors that couple to phospholipase C’s ( PLC’s). As expected, outward NCX1 current (i.e. Ca influx) can be strongly inhibited when M1 agonists induce PIP2 depletion. However, inward currents (i.e. Ca extrusion) without cytoplasmic Na can be increased markedly in parallel with an increase of cell capacitance (i.e. membrane area). Similar effects are incurred by cytoplasmic perfusion of GTP or the actin cytoskeleton disruptor, latrunculin, even in the presence of nonhydrolyzable ATP (AMP-PNP). Thus, G-protein signaling may increase NCX1 currents by destabilizing membrane cytoskeleton-PIP2 interactions. Second, to increase PIP2 we directly perfused PIP2 into cells. Outward NCX1 currents increase as expected. But over minutes currents decline substantially, and cell capacitance usually decreases in parallel. Third, using BHK cells with stable NCX1 expression, we increased PIP2 by transient expression of a phosphatidylinositol-4-phosphate-5-kinase (hPIP5KI) and a PI4-kinase (PI4KII). NCX1 current densities were decreased by >80 and 40%, respectively. Fourth, we generated transgenic mice with 10-fold cardiac-specific overexpression of PI4KII. This wortmannin-insensitive PI4KII was chosen because basal cardiac phosphoinositides are nearly insensitive to wortmannin, and surface membrane PI4-kinase activity, defined functionally in excised patches, is not blocked by wortmannin. Both PIP and PIP2 were increased significantly, while NCX1 current densities were decreased by 78% with no loss of NCX1 expression. Most mice developed cardiac hypertrophy, and immunhistochemical analysis suggests that NCX1 is redistributed away from the outer sarcolemma. Cholera toxin uptake was increased three-fold, suggesting that clathrin-independent endocytosis is enhanced. We conclude that direct effects of PIP2 to activate NCX1 can be strongly modulated by opposing mechanisms in intact cells that probably involve membrane cytoskeleton remodeling and membrane trafficking.