The effects of caffeine on extracellular dopamine and acetylcholine have been studied in freely moving rats implanted with concentric microdialysis probes in the nucleus accumbens shell and core and in the medial prefrontal cortex. Intravenous administration of caffeine (0.25, 0.5, 1.0, 2.5 and 5.0 mg/kg) dose-dependently increased dopamine and acetylcholine dialysate concentrations in the medial prefrontal cortex, while it did not affect dialysate dopamine in the shell and core of the nucleus accumbens. Intraperitoneal administration of caffeine (1.5, 3, 10, 30 mg/kg) also failed to affect DA in the shell and core of the nucleus accumbens. Such effects were duplicated by intravenous administration of DPCPX, a selective antagonist of adenosine A1 receptors, and of SCH 58261, an antagonist of A2a receptors. The effect of caffeine on prefrontal dopamine and acetylcholine transmission was also studied in rats chronically administered with caffeine (25 mg/kg, twice a day for seven days). At the end of this treatment rats became tolerant to the locomotor stimulating effects of a dose of 1 and 2.5 mg/kg i.v. of caffeine; these doses, however, still increased dialysate acetylcholine but did not affect dopamine in the prefrontal cortex. Therefore, in rats made tolerant to the locomotor stimulant effects of caffeine, tolerance developed to the dopamine stimulant but not to the acetylcholine stimulant effect of caffeine in the prefrontal cortex. The lack of acute stimulation of dopamine release in the nucleus accumbens shell by caffeine is relevant to the issue of its addictive properties and of the role of DA in drug- and substance-addiction. On the other hand, the dissociation between tolerance to the locomotor effects of caffeine and stimulation of acetylcholine release in the prefrontal cortex suggests that this effect might be correlated to the arousing effects of caffeine as distinct from its locomotor stimulant properties.