The interior of purified cholinergic Torpedo vesicles is acidic, pHin = 5.2 at external pH = 7.4. The internal pH changes linearily as a function of external pH yielding ΔpH = 2.0 and 2.5 at pHout = 6.3 and 9.1 respectively. The proton translocator carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) + the ionophore valinomycin dissipate the proton gradient across the vesicular membrane and concurrently induce acetylcholine release from vesicles suspended in K+ buffer. The effect of FCCP + valinomycin is not sensitive to external pH values between 6.3 and 9.1 and is diminished at lower external pH. The possible role of intravesicular pH and of the proton electrochemical gradient in the storage of acetylcholine within cholinergic vesicles is discussed.