A Whole-cell patch-clamp study was carried out to elucidate modulatory roles of serotonin (5-HT) in GABAergic transmission onto basal forebrain cholinergic neurons, using brain slice preparations obtained from P12-19 rat under deep anesthesia with halothane. Animal handling and all experimental procedures were done according to the Guiding Principles for the care and Use of Animals in the Field of Physiological Sciences of the Physiological Society of Japan (2009), the UK Animals (Scientific Procedures) Act 1986, and the guidelines of Jikei University School of Medicine. GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) were evoked by focal electrical stimulation in the presence of CNQX, D-AP5 and strychnine to block non-NMDA-, NMDA- and glycine receptor-mediated currents components, respectively. Bath application of 5-HT reduced the amplitude of the evoked GABAergic IPSCs in a concentration-dependent manner between 0.1-100 μM The inhibition of IPSCs by 0.1, 1, 10 and 100 μM 5-HT was 21.1 ± 9.17 % (n=6), 35.2 ± 5.52 % (n=5), 58.5 ± 2.36 % (n=41) and 64.1 ± 5.46 % (n=4), respectively. A 5-HT1B receptor agonist, CP93129 (10 μM) also suppressed the IPSCs by 59.8 ± 9.25 % (n=5), whereas a 5-HT1A receptor agonist, 8-OH-DPAT, had no effect. NAS-181, a 5-HT1B receptor antagonist antagonized the 5-HT-induced suppression of IPSCs, whereas NAN-190, a 5-HT1A receptor antagonist did not. 5-HT (1 μM) reduced the frequency of spontaneous miniature IPSCs (mIPSCs) by 52.0 ± 9.85 % (n=6) without changing their amplitude distribution. This effect remained unchanged when extracellular Ca2+ was replaced by Mg2+ (5 mM). In the presence of ω-Aga-TK, ω-CgTX, or SNX-482, an N-, P/Q- or R-type calcium channel blocker, respectively, 5-HT could still inhibit the IPSCs. These findings suggest that activation of presynaptic 5-HT1B receptors on the terminals of GABAergic afferents to basal forebrain cholinergic neurons inhibits GABA release in Ca2+-independent manner.