Previous studies have shown that dopamine (DA) may play an important role in mediating or modulating the facilitating action of clozapine in glutamatergic transmission. This possibility was tested further in the present study by pharmacological manipulation of the DA system. When rats were pretreated with reserpine (which blocks storage of biogenic amines) and alpha-methyl para-tyrosine (AMPT, which inhibits tyrosine hydroxylase, the rate-limiting enzyme for the DA synthesis), the ability of clozapine to augment glutamatergic transmission in pyramidal cells of the medial prefrontal cortex (mPFC) was totally abolished. Furthermore, the application of l-dihydroxyphenylalanine (L-DOPA, the immediate precursor of DA which bypasses the synthesis step inhibited by AMPT) reversed the effect produced by reserpine plus AMPT and re-instated the facilitating action of clozapine, whereas administration of 5-hydroxytryptophan (5-HTP), the immediate precursor of 5-HT, was ineffective. In addition, DA D1 receptor antagonist SCH 23390 also completely prevented clozapine-induced facilitating action in the mPFC pyramidal cells. The present results demonstrate that newly synthesized DA and DA D1 receptors are required for clozapine to elicit its facilitating action on glutamatergic neurotransmission in the mPFC.