We investigated regulation of the type 1 isoform of the Na+/H+ exchanger by phosphorylation. Four specific groups of serine and threonine residues in the regulatory carboxyl terminal tail were mutated to alanine residues; #1, S693A; #2, T718A,S723/726/729A; #3, S766/770/771A; and #4, T779A,S785A. The proteins were expressed in Na+/H+ exchanger-deficient cells and the activity was characterized. All mutants had proper expression, localization and normal basal activity relative to wild type NHE1. Sustained intracellular acidosis was used to activate NHE1 via an ERK-dependent pathway, that could be blocked with the MEK inhibitor U0126. Immunoprecipitation of [32-P] labeled Na+/H+ exchanger from intact cells showed that sustained intracellular acidosis increased Na+/H+ exchanger phosphorylation in vivo. This was blocked by U0126. The Na+/H+ exchanger activity of mutants 1 and 2 was stimulated similar to wild type Na+/H+ exchanger. Mutant 4 showed a partially reduced level of activation. However, mutant #3 was not stimulated by sustained intracellular acidosis and loss of stimulation of activity correlated to a loss of sustained acidosis-mediated phosphorylation in vivo. Mutation of the individual amino acids within mutant #3, Ser766, Ser770 and Ser771, showed that Ser770 and Ser 771 are responsible for mediating increases in NHE1 activity through sustained acidosis. Both intact Ser770 and Ser771 were required for sustained acidosis-mediated activation of NHE1. Our results suggest that amino acids Ser770 and Ser771 mediate ERK-dependent activation of the Na+/H+ exchanger in vivo.