Blockade of NMDA/glutamate receptors induces altered behavior in humans and experimental animals. At the same time a differential activation of dopaminergic (DA) systems has been reported. To study the involvement of the medial prefrontal cortex (mPFC) in these effects, we used bilateral perfusions of the rat mPFC with the competitive NMDA-antagonist D-AP-5 and simultaneous determination of spontaneous behavior and local DA efflux. D-AP-5 concentration-dependently induced arousal and motor activity and also increased DA efflux. These effects were shown to have a similar time-scale but no causal relationship: combined D1/D2 receptor blockade in the mPFC did not inhibit the behavioral activation. As bilateral perfusion of the nucleus accumbens with D-AP-5 resulted in similar behavioral effects, but no change in DA efflux, we conclude that DA is not involved in the behavioral activation induced by these local perfusions. However, local blockade of non-NMDA glutamate receptors or stimulation of GABA-B receptors completely blocked the effects on behavior and DA efflux, suggesting that the arousal and locomotor activity induced by NMDA receptor blockade in mPFC is primarily dependent on activation of glutamatergic mechanisms. The mPFC appears to be an important site of action for NMDA antagonists to induce behavioral alterations.