In situ hybridization histochemistry and in vivo microdialysis were combined to study the effect of amphetamine on the expression of choline acetyltransferase and glutamate decarboxylase67 mRNA and in vivo release of acetylcholine and GABA in rat medial nucleus accumbens. Differential effects on acetylcholine and GABA neurons by a single challenge injection of amphetamine (1.5 mg/kg, s.c.) were apparent in saline-pretreated and amphetamine-pretreated (same dose, twice daily for the previous seven days) rats. Extracellular acetylcholine levels were increased up to 50% over a prolonged period following both single and repeated amphetamine. In contrast, extracellular concentrations of GABA were gradually decreased to half the control values, but only in rats receiving repeated amphetamine. Although the increase of acetylcholine release was not associated with any change in choline acetyltransferase mRNA levels, the number of neurons expressing high levels of glutamate decarboxylase67 mRNA was decreased (28%) following repeated injections. Thus we suggest that amphetamine decreases extracellular GABA levels by a slow mechanism, associated with the decreased expression of glutamate decarboxylase67 mRNA in a subpopulation of densely labeled neurons in the medial nucleus accumbens. The delayed response by GABA to amphetamine may reflect supersensitivity in the activity of postsynaptic gamma-aminobutyric acid-containing neurons in nucleus accumbens as a consequence of the repeated amphetamine treatment.