In Ehrlich ascites tumor cells, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) acts at two different sites depending upon the concentration employed. (1) In non-glycolysing respiring cells, FCCP is seen to uncouple the mitochondria and thereby it inhibits the ATP-dependent (Na+, K+) pump. (2) In glycolysing cells, FCCP does not affect the electrogenic (Na+, K+) pump, but depolarizes the plasma membrane potential difference as visualized by the distribution of the lipid-soluble cation, tetraphenylphosphonium, and by an inhibition of the rheogenic, Na+-dependent uptake of alpha-aminoisobutyric acid. A depolarization by FCCP also occurs under conditions where a K+-diffusion potential is present and the pump is blocked by metabolic inhibition or by ouabain. Depolarization and FCCP-induced increase in H+ fluxes across the plasma membrane exhibit a similar FCCP-concentration dependency. The imposition of proton-concentration differences in the presence of FCCP inhibits (pHi > pHo) or stimulates (pHi < pHo) alpha-aminoisobutyric acid uptake and tetraphenylphosphonium accumulation. The experiments indicate that FCCP shifts the plasma membrane potential of Ehrlich cells, which are normally relatively impermeable for protons, towards an H+-diffusion potential.