2-(4-Phenylpiperidino)cyclohexanol (AH-5183) and 2-bromo-alpha-ergocryptine, known inhibitors of the transport of acetylcholine and L-glutamate, respectively, into synaptic vesicles, inhibited the ATP-dependent uptake of dopamine in parallel with the dissipation of the electrochemical gradient of protons in chromaffin granule membrane vesicles. These compounds induced the release of accumulated dopamine from the vesicles. They also inhibited the ATP-dependent formation of the electrochemical gradient of protons in liposomes reconstituted with chromaffin H(+)-ATPase without affecting the activities for ATP hydrolysis, and ATP-dependent uptakes of dopamine, gamma-aminobutyrate, and glutamate into synaptic vesicles. These results indicated that 2-(4-phenylpiperidino)cyclohexanol and 2-bromo-alpha-ergocryptine acted as uncouplers in the secretory vesicles.