Activation of γ-aminobutyric acid B (GABAB) and 5-hydroxytryptamine (5-HT) receptors produces presynaptic inhibition at glutamatergic terminals in the rat neocortex. To evaluate interactions between these metabotropic receptors, field potentials were recorded in layer 2/3 of somatosensory cortex. In addition, the paired pulse (PP) protocol was used to measure changes in the ratio of the second/first extracellular synaptic potentials (S2/S1 ratio) as an index of glutamate release probability in the area. Lowering extracellular [Ca2+]o to 0.5 mM, increased the S2/S1 ratio by 318 ± 134%. 5-HT (1 μM) increased the S2/S1 ratio by 61 ± 15%. In presence of the GABAA antagonist bicuculline (10 μM), 5-HT increased the S2/S1 ratio by 98 ± 15%. This effect did not desensitize after two consecutive applications of the amine, and was dose dependent in the concentration range between 0.03–1 μM (EC50 = 2.36 × 10−7 mol/L). The increase of S2/S1 ratio induced by 5-HT (1 μM) was blocked reversibly by the 5-HT1A antagonist NAN-190 (10–30 μM), but was unaffected by the selective GABAB antagonist CGP 52432 (1 μM). The action of 5-HT was mimicked by the 5-HT1A/7 agonist 8OH-DPAT (10 μM), increasing the S2/S1 ratio by 84 ± 2%, a response that was unaffected by the 5-HT2/7 antagonist ritanserin (2 μM). The 5-HT1B agonist CP93129 (10 μM) had no effect. The GABAB agonist baclofen (1 μM) increased the S2/S1 ratio up to 308 ± 33%, which is similar to that produced by low [Ca2+]o. When the effect of baclofen was maximal, application of 5-HT (1 μM) reversed the S2/S1 ratio back to 78 ± 27%, a result that was blocked by the 5-HT2/7 antagonist ritanserin (100 nM). Notably, the interaction between the GABAB agonist and 5-HT was order dependent, because enhancement of the S2/S1 ratio elicited by baclofen was not inhibited if 5-HT was applied first. These results suggest a complex interaction between GABAB, 5-HT1A, and 5-HT2 receptors in layer 2/3 of rat somatosensory cortex. Activation of GABAB receptors induces PP facilitation (inhibits glutamate release) more efficiently than does activation of 5-HT1A receptors. When the effect of GABAB receptor activation is maximal, however, the influence of 5-HT changes to the opposite direction, inhibiting PP facilitation (increasing glutamate release) through activation of 5-HT2 receptors. © 2004 Wiley-Liss, Inc.