The techniques of intracellular recording and single-electrode voltage-clamp were used to study the effect of serotonin (5-HT) and the selective 5-HT3 receptor agonist SR 57227A on N-methyl-D-aspartic acid (NMDA)-evoked responses in pyramidal cells of the rat medial prefrontal cortex (mPFC) in in vitro brain slice preparations. Bath application of 5-HT or SR 57227A produced a concentration-dependent inhibition of NMDA-induced membrane depolarization, action potentials, and inward current. The depressant action of 5-HT and SR 57227A had a slow onset and showed no signs of receptor desensitization. This action was markedly attenuated or completely blocked by the selective 5-HT3 receptor antagonists granisetron and BRL 46470A, but not other receptor antagonists. In addition to inhibiting NMDA-evoked responses, SR 57227A also depressed significantly pharmacologically isolated, NMDA receptor-mediated, monosynaptic excitatory postsynaptic currents (EPSCs) elicited by electrical stimulation of the forceps minor; this inhibitory action was blocked by BRL 46470A but not other 5-HT receptor antagonists. Perfusion of Ca2+-free or low Ca2+ plus Cd2+ artificial cerebrospinal fluid prevented electrical stimulation-induced EPSCs, but did not affect the inhibitory action of 5-HT and SR 57227A. In conclusion, we demonstrate for the first time that 5-HT and SR 57227A interact with 5-HT3-like receptors to produce a direct inhibitory action on NMDA receptor-mediated response in pyramidal cells of the mPFC.