A whole-cell patch-clamp study was carried out to elucidate serotonin (5-HT)-induced modulation of excitatory synaptic transmission onto cholinergic neurons in the basal forebrain (BF) using brain slices obtained from young rats (P12-20). BF cholinergic neurons were identified with Cy3-192IgG. Excitatory postsynaptic currents (EPSCs) were evoked by focal stimulation. 5-HT, 8-OH-DPAT (DPAT), a 5-HT_1A receptor agonist or CP93129 (CP), a 5-HT_1B receptor agonist inhibited the amplitude of EPSCs. 5-HT-induced inhibition was mostly antagonized in the presence of both 5-HT_1A and 5-HT_1B receptor antagonists. Paired-pulse ratio (PPR) and coefficient of variation (CV) of the EPSCs were increased by CP, whereas DPAT had no effect on PPR or CV. DPAT inhibited the inward currents induced by puff application of L-glutamate, whereas CP had no effect. 5-HT-induced inhibition was decreased in the presence of ω-agatoxin TK (Aga) compared to that without Aga. Furthermore, CP-induced inhibition of EPSCs was eliminated in the presence of Aga, whereas DPAT still inhibited the EPSCs even in the presence of Aga. These results suggest that 5-HT_1A receptors reduce the sensitivity of postsynaptic glutamate receptors, whereas 5-HT_1B receptors presynaptically inhibit glutamate release by selectively blocking P/Q-type calcium channels, thereby both inhibiting excitatory transmission onto BF cholinergic neurons.