Amphetamine-like psychostimulants are thought to produce rewarding effects by increasing dopamine levels at mesolimbic synapses. Paradoxically, dopamine uptake blockers, which generally increase extracellular dopamine, inhibit amphetamine-induced dopamine overflow. This effect could be due to either inhibition of amphetamine uptake or inhibition of dopamine efflux through the transporter (reverse transport). We used weak bases and dopamine uptake blockers in ventral midbrain neuron cultures to separate the effects on blockade of amphetamine uptake from reverse transport of dopamine. Amphetamine, ammonium chloride, tributylamine, and monensin, at concentrations that produce similar reductions in acidic pH gradients, increased dopamine release. This effect was inhibited by uptake blockers. Although in the case of amphetamine the inhibition of release could have been due to blockade of amphetamine uptake, inhibition also occurred with weak bases that are not transporter substrates. This suggests that reduction of vesicular pH gradients increases cytoplasmic dopamine which in turn promotes reverse transport. Consistent with this model, extracellular 3,4-dihydroxyphenylacetic acid was increased by ammonium chloride and monensin, as would be expected with elevated cytoplasmic dopamine levels. These findings extend the weak base mechanism of amphetamine action, in which amphetamine reduces vesicular pH gradients resulting in increased cytoplasmic dopamine that promotes reverse transport.