Activity of the Na؉ /H ؉ exchanger (NHE) isoform 1 (NHE1) is increased by intracellular acidosis through the interaction of intracellular H ؉ with an allosteric modifier site in the transport domain. Additional regulation is achieved via kinase-mediated modulation of the NHE1 regulatory domain. To determine if intracellular acidosis stimulates NHE1 activity solely by the allosteric mechanism, we subjected cultured neonatal rat ventricular myocytes (NRVM) with native NHE1 expression to intracellular acidosis (pH i ϳ 6.6) for up to 6 RSK and abolished the stimulation of NHE activity by sustained (3 min) intracellular acidosis. Our data show that not only the extent but also the duration of intracellular acidosis regulates NHE1 activity and suggest that the stimulatory effect of sustained intracellular acidosis occurs through a novel mechanism mediated by activation of the ERK pathway.