The dorsal (DR) and median (MR) raphe nuclei contain 5-hydroxytryptamine (5-HT) cell bodies that give rise to the majority of the ascending 5-HT projections to the forebrain. The DR and MR have differential roles in mediating stress, anxiety and depression. Glutamate and GABA activity sculpt putative 5-HT neuronal firing and 5-HT release in a seemingly differential manner in the MR and DR, yet isolated glutamate and GABA activity within the DR and MR has not been systematically characterized. Visualized whole-cell voltage-clamp techniques were used to record excitatory and inhibitory postsynaptic currents (EPSC and IPSC) in 5-HT-containing neurons. There was a regional variation in action potential-dependent (spontaneous) and basal [miniature (m)] glutamate and GABAergic activity. mEPSC activity was greater than mIPSC activity in the DR, whereas in the MR the mIPSC activity was greater. These differences in EPSC and IPSC frequency indicate that glutamatergic and GABAergic input have distinct cytoarchitectures in the DR and MR. 5-HT(1B) receptor activation decreased mEPSC frequency in the DR and the MR, but selectively inhibited mIPSC activity only in the MR. This finding, in concert with its previously described function as an autoreceptor, suggests that 5-HT(1B) receptors influence the ascending 5-HT system through multiple mechanisms. The disparity in organization and integration of glutamatergic and GABAergic input to DR and MR neurons and their regulation by 5-HT(1B) receptors may contribute to the distinction in MR and DR regulation of forebrain regions and their differential function in the aetiology and pharmacological treatment of psychiatric disease states.