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To precisely identify the cytoplasmic subdomains that are responsible for the intracellular pH (pH1)-sensitivity, ATP depletion-induced inhibition and Ca2+ activation of the Na+/H+ exchanger (NHE1), we generated a set of deletion mutants of carboxyl-terminal cytoplasmic domain and expressed them in the exchanger-deficient cell line PS120. We evaluated pH1-sensitivity of these mutants by measuring the resting pH1 in cells placed in an acidic medium (pH 6.0) and pH1-dependence of 5-(N-ethyl-N-isopropyl)amiloride–sensitive 22Na+ uptake. Detailed analysis revealed that the cytoplasmic domain of NHE1 is consists of at least four subdomains in terms of pH1-sensitivity of the unstimulated NHE1:I, aa 516–590/595; II, aa 596–635; III, aa 636–659; and IV, aa 660–815. Subdomains II and IV were silent for pH1-sensitivity. Subdomain I had a pH1-maintenance function, preserving pH1-sensitivity in a physiological range, whereas subdomain III, overlapping with the high affinity calmodulin (CaM)-binding site, exhibited an autoinhibitory function. Deletion of subdomain I abolished the decrease of pH1-sensitivity induced by cell ATP depletion, indicating that domain I plays a crucial role in this phenomenon. Deletion of subdomain III rendered the inhibition by ATP depletion less efficient, suggesting the possible interaction between subdomains I and III. On the other hand, tandem elongation of subdomain II by insertion did not affect either the inhibitory function of domain III or the removal of this inhibition by ionomycin or thrombin. However, deletion of subdomain II partially abolished the inhibitory effect of subdomain III. Subdomain II thus seems to function as a mobile “flexible loop,” permitting the CaM-binding subdomain III to exert its normal function. These findings, together with our previous data, support a concept that cell ATP, Ca2+, and growth factors regulate NHE1 via a mechanism involving direct or indirect interactions of specific cytoplasmic subdomains with the “H+-modifier site.” [ABSTRACT FROM AUTHOR]