Na+i-dependent Ca2+ uptake, Na+-dependent Ca2+ release, and PtdIns-4,5-P2 binding to Na+/Ca2+ exchanger (NCX1) as a function of extravesicular (intracellular) [Ca2+] were measured. Alkalinization increases Ca2+i affinity and PtdIns-4,5-P2 bound to NCX1; these effects are abolished by pretreatment with PtdIns-PLC and are insensitive to MgATP. Acidification reduces Ca2+i affinity. MgATP reverts it only partially despite the fact that the PtdIns-4,5-P2 bound to NCX1 reaches the same levels as at pH 7.8. Extravesicular Na+-stimulated and Ca2+-dependent Ca2+efflux indicate the Ca2+ regulatory site is involved. Therefore, to display maximal affinity to Ca2+i, PtdIns-4,5-P2 binding and deprotonation of NCX1 are simultaneously need.