The possibility that the dopaminergic neurons innervating the medial prefrontal cortex (mPFC) can inhibit locomotor behaviour has been suggested in several studies. The evidence remains indirect, however, because the manipulations tested aimed exclusively at permanently depleting mPFC dopamine. Here we demonstrate in rats that acute increases in dopamine transmission in this site by local injections of amphetamine inhibit the known locomotor-activating effects of amphetamine in the nucleus accumbens (N.Acc). Further, intra-mPFC injections of the D-1 dopamine receptor antagonist SCH-23390, but not other dopamine antagonists with greater affinities for noradrenergic, serotonergic and D-2 dopamine receptors, enhanced the locomotion induced by intra-N.Acc. amphetamine. These findings provide direct evidence for the inhibition of locomotor activity by mPFC dopamine and suggest that it is acting at D-1 dopamine receptors in this site.