The modulatory roles of serotonin in non-NMDA gulutamatergic transmission onto basal forebrain cholinergic neurons were investigated in P12-20 rat brain slices using whole-cell patch-clamp technique. Basal forebrain cholinergic neurons were identified with Cy3-192IgG injected into lateral ventricles 3-7 prior to the experiments. Pharmacologically isolated non-NMDA receptor-mediated excitatory postsynaptic currents were evoked by focal electrical stimulation. Bath application of serotonin at 10 or 30 μM inhibited the amplitude of the evoked excitatory postsynaptic currents by 28.1 ± 6.26% (n=6) and 41.0 ± 10.7% (n=6), respectively. A 5-HT_1A receptor agonist, (R)-(+)-8-OH-DPAT (30 μM), and a 5-HT_1B receptor agonist, CP93129 (30 μM), both also suppressed the excitatory postsynaptic currents. 5-HT-induced suppression of excitatory postsynaptic currents is significantly larger in low (1.0 mM) Ca²⁺-containing external solution, compared with in normal (2.4 mM Ca²⁺) Krebs solution. These results suggest that 5-HT inhibits non-NMDA glutamatergic transmission onto basal forebrain cholinergic neurons via 5-HT_1A and 5-HT_1B receptors, and that the inhibitory effect is saturated in the normal external solution.