Phosphatidylinositol biphosphate (PtdIns-4,5P 2 ) plays a key role in the regulation of the mammalian heart Na + /Ca 2+ exchanger (NCX1) by protecting the intracellular Ca 2+ regulatory site against H + i and (H + i +Na + i ) synergic inhibition. MgATP and MgATP-γ-S up-regulation of NCX1 takes place via the production of this phosphoinositide. In microsomes containing PtdIns-4,5P 2 incubated in the absence of MgATP and at normal [Na + ] i , alkalinization increases the affinity for Ca 2+ i to the values seen in the presence of the nucleotide at normal pH; under this condition, addition of MgATP does not increase the affinity for Ca 2+ i any further. On the other hand, prevention of Na + i inhibition by alkalinization in the absence of MgATP does not take place when the microsomes are depleted of PtdIns-4,5P 2 . Experiments on NCX1–PtdIns-4,5P 2 cross-coimmunoprecipitation show that the relevant PtdIns-4,5P 2 is not the overall membrane component but specifically that tightly attached to NCX1. Consequently, the highest affinity of the Ca 2+ i regulatory site is seen in the deprotonated and PtdIns-4,5P 2 -bound NCX1. Confirming these results, a PtdIns-5-kinase also cross-coimmunoprecipitates with NCX1 without losing its functional competence. These observations indicate, for the first time, the existence of a PtdIns-5-kinase in the NCX1 microdomain.