Using bovine heart sarcolemma vesicles we studied the effects of protons and phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) on the affinity of the mammalian Na+/Ca2+ exchanger (NCX1) for intracellular Ca2+. By following the effects of extravesicular ligands in inside-out vesicles, their interactions with sites of NCX1 facing the intracellular medium were investigated. Two Na+-gradient-dependent fluxes were studied: Ca2+ uptake and Ca2+ release. PtdIns-4,5-P2 binding to NCX1 was investigated in parallel. Without MgATP (no ‘de novo’ synthesis of PtdIns-4,5-P2), alkalinization increased the affinity for Ca2+ and the PtdIns-4,5-P2 bound to NCX1. Vesicles depleted of phosphoinositides were insensitive to alkalinization, but became responsive following addition of exogenous PtdIns-4,5-P2 or PtdIns plus MgATP. Acidification reduced the affinity for Ca2+ev; this was only partially reversed by MgATP, despite the increase in bound PtdIns-4,5-P2 to levels observed with alkalinization. Inhibition of Ca2+ uptake by increasing extravesicular [Na+] indicates that it is related to Hqi and Naqi synergistic inhibition of the Ca2+i regulatory site. Therefore, the affinity of the NCX1 Ca2+i regulatory site for Ca2+ was maximal when both intracellular alkalinization and an increase in PtdIns-4,5-P2 bound to NCX1 (not just of the total membrane PtdIns-4,5-P2) occurred simultaneously. In addition, protons influenced the distribution, or the exposure, of PtdIns-4,5-P2 molecules in the surroundings and/or on the exchanger protein.