SummaryIntracellular pH must be kept close to neutrality to be compatible with cellular functions, but the mechanisms of pH homeostasis and the responses to intracellular acidification are mostly unknown. In the plantArabidopsis thaliana, we found that intracellular acid stress generated by weak organic acids at normal external pH induces expression of several chaperone genes, includingROF2, which encodes a peptidyl‐prolylcis‐transisomerase of the FK506‐binding protein class. Loss of function ofROF2, and especially double mutation ofROF2and the closely related geneROF1, results in acid sensitivity. Over‐expression ofROF2confers tolerance to intracellular acidification by increasing proton extrusion from cells. The activation of the plasma membrane proton pump (H+‐ATPase) is indirect: over‐expression ofROF2activates K+uptake, causing depolarization of the plasma membrane, which activates the electrogenic H+pump. The depolarization ofROF2over‐expressing plants explains their tolerance to toxic cations such as lithium, norspermidine and hygromycin B, whose uptake is driven by the membrane potential. AsROF2induction and intracellular acidification are common consequences of many stresses, this mechanism of pH homeostasis may be of general importance for stress tolerance.