The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH in the myocardium. NHE1 is also important in mediating myocardial hypertrophy and blockage of NHE1 activity prevents hypertrophy and reduces ischemia-reperfusion injury in animal models. We recently demonstrated that extracellular regulated kinase (ERK) mediated activation of NHE1 occurs during ischemia-reperfusion of the myocardium. To understand regulation of NHE1 in the myocardium by phosphorylation, we expressed a series of adenoviruses that express wild type and mutant cDNA for NHE1. All exogenous cDNA for NHE1 had additional mutations [Leu163Phe/Gly174Ser] which increases NHE1 resistance to EMD87580 (a specific blocker of NHE1) 100-fold and allowed measurement of exogenous NHE1 while inhibiting endogenous NHE1. By examining the effects of a series of mutations of the NHE1 cytosolic region, we determined that amino acids Ser⁷⁷⁰ and Ser⁷⁷¹ were essential for acute activation of NHE1 activity in rat cardiomyocytes. Specific mutation of either residue prevented rapid activation of exchanger activity by sustained intracellular acidosis through ERK-dependent pathways. The same amino acids were critical to phenylephrine-mediated, ERK-dependent activation of NHE1 activity and increased phosphorylation in intact rat cardiomyocytes. The results demonstrate that both sustained intracellular acidosis and phenyephrine rapidly activate the NHE1 protein in intact cardiac cells through ERK-dependent pathways that act on a common pathway mediated by amino acids Ser⁷⁷⁰ and Ser⁷⁷¹ of the cytosolic tail of the protein.