Eukaryotic cells constantly have to fight against internal acidification. In mammals, this task is mainly performed by the ubiquitously expressed electroneutral Na(+)/H(+) exchanger NHE-1, which activates in a cooperative manner when cells become acidic. Despite its biological importance, the mechanism of this activation is still poorly understood, the most commonly accepted hypothesis being the existence of a proton-sensor site on the internal face of the transporter. This work uncovers mutations that lead to a nonallosteric form of the exchanger and demonstrates that NHE-1 activation is best described by a Monod-Wyman-Changeux concerted mechanism for a dimeric transporter. During intracellular acidification, a low-affinity form of NHE-1 is converted into a form possessing a higher affinity for intracellular protons, with no requirement for an additional proton-sensor site on the protein. This new mechanism also explains the activation of the exchanger by growth signals, which shift the equilibrium towards the high-affinity form.