The effect of the protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) was studied on the intracellular [Na+], pH, and plasma membrane potential in isolated nerve terminals. FCCP induced a rise of [Na+]i at, and even below, the concentrations (0.025-1 microM) in which it is usually used in intact cells to eliminate Ca2+ uptake by mitochondria. The FCCP-induced increase of [Na+]i correlates with a fall in both the ATP level and the ATP/ADP ratio. In addition, a sudden rise of the intracellular proton concentration ([H+]i) from 83 +/- 0.4 to 124 +/- 0.7 nM was observed on the addition of FCCP (1 microM). Parallel with the rise in [H+]i, an abrupt depolarization was detected, followed by a slower decrease in the plasma membrane potential. Both the extent of the pHi change and the fast depolarization of the plasma membrane were proportional to the proton electrochemical gradient across the plasma membrane; when this gradient was increased, greater depolarization was detected. The slower decrease of the membrane potential after the fast initial depolarization was abolished when the medium contained no Na+. It is concluded that FCCP (1) gives rise to a depolarization by setting the plasma membrane potential close to the proton equilibrium potential and (2) enhances the intracellular [Na+] as a consequence of an insufficient ATP level and ATP/ADP ratio to fuel the Na+,K+/ATPase. Because both disturbed Na+ homeostasis and plasma membrane depolarization could profoundly interfere with Ca2+ homeostasis in the presence of protonophores, consideration given to these alterations may help to clarify the cellular Ca2+ sequestration processes.