Serotonin [5-hydroxytryptamine (5-HT)] plays many important roles as a neuromodulator in the central nervous system. In our previous studies, 5-HT inhibited the induction of long-term potentiation (LTP) and depression (LTD) of layer 2/3 field potentials evoked by stimulation of layer 4 in rat visual cortical slices. To study the underlying mechanisms, we investigated the effect of 5-HT on the synaptic transmission mediated by the three major neurotransmitter receptors: AMPA receptors, NMDA receptors, and GABAA receptors. Whole-cell patch-clamp recording was made on the pyramidal neurons of layer 2/3. AMPA and NMDA receptors currents were measured with K-gluconate-based internal solution. GABAA receptor current was measured with CsCl-based internal solution. AMPA receptor current was measured with a NMDA receptor blocker at -70 mV of holding potential. NMDA receptor current was measured with AMPA and GABAA receptor blockers at 0 mV of holding potential. GABAA receptor current was measured with AMPA and NMDA receptors blockers at -75 mV of holding potential. To study the effect of 5-HT on long-term synaptic plasticity, LTD of excitatory synaptic potential was induced with low frequency stimulation (1 Hz, 900 stimulation) of the paired pulse stimuli (40-ms interval). Stimulation was applied to either layer 1 (L1-LTD) or layer 4 (L4-LTD). In both stimulation pathways, LTD was well induced. The induction of L1-LTD depended on both metabotropic glutamate and NMDA receptors. However, only NMDA receptor was involved in L4-LTD. Application of 5-HT inhibited L4-LTD but not L1-LTD. The most significant effect of 5-HT on synaptic transmission was ~20% increase in the GABAA receptor current evoked by layer 4 stimulation only. To test the importance of this increase on the suppression of LTD, we investigated the effect of GABAA receptor modulators with concentration at which GABAA receptor current increase or decrease by ~20%. The 20% increase in GABAA receptor current was sufficient to block the NMDA receptor-dependent L4-LTD but could not block L1-LTD. The 20% decrease in GABAA receptor current was sufficient to reverse the inhibitory effect of 5-HT on L4-LTD. These results suggest that inhibitory role of 5-HT on NMDA receptor-dependent LTD might be mediated by the enhancement of GABAA receptor current which could suppress the activation of NMDA receptors. The enhancing effect of 5-HT on GABAA receptor current was input-specific and the specific input depended heavily on NMDA receptors for the induction of LTD.