The inhibitory effects of 5-hydroxytryptamine (5-HT) on interhemispheric and intracortical synaptic potentials in layer V neurons of the rat medial prefrontal (MFC) cortex were examined. Low concentrations (1-3 microM) of 5-HT selectively attenuated polysynaptic potentials that were similarly evoked by callosal or white matter stimulation. Maximally effective concentrations of 5-HT blocked interhemispheric transmission by 50-90%, as evidenced by an attention of the short latency callosal depolarizing synaptic potential (e-DPSP). These effects of 5-HT were not associated with a change in membrane potential or input resistance. The e-DPSP was characterized as having an N-methyl-D-aspartate (NMDA) and a non-NMDA component; the non-NMDA component was attenuated by 5-HT. Attenuation of the synaptic potential was accompanied by an attenuation of a postsynaptic glutamate potential. Suppression of both the e-DPSP and the glutamate potential was concentration dependent with 10-100 microM being maximally effective. The 5-HT1A/2 antagonist, spiperone, antagonized the effects of 5-HT on synaptic and glutamate potentials. Spiperone (1 microM) shifted the concentration-effect curves for suppression of the e-DPSP and the glutamate potential to the right; however, the Kb for the glutamate potential concentration-effect curve was 10 times that for the e-DPSP curve. The differential antagonist sensitivity of synaptic and glutamate potentials was an indication that serotonin acted on more than one receptor subtype to reduce interhemispheric transmission.