Intracellular recordings were made from neurons of the nucleus prepositus hypoglossi in slices of guinea pig medulla. 5-HT (serotonin) caused a hyperpolarization followed by a late depolarization. The hyperpolarization was mediated by 5-HT1A receptor activation and could be selectively blocked by pindobind-5HT1A (PBD). 5-HT then caused a depolarization only. A selective 5-HT2 agonist, (+)-1-(2,5-dimethoxy-4- iodophenyl)-2-aminopropane (DOI), also caused a depolarization. Ketanserin and spiperone, 5-HT2 antagonists, blocked the depolarization due to both 5-HT and DOI. Focal electrical stimulation caused an IPSP mediated by 5-HT acting upon 5-HT1A receptors and a slow EPSP (s-EPSP). PBD blocked the IPSP, leaving an isolated s-EPSP. Both spiperone and ketanserin antagonized the s-EPSP, while DOI occluded it. The s-EPSP was from 2 to 10 mV in amplitude and 35–50 sec in duration, and showed voltage dependence consistent with a decrease in potassium conductance. Both the IPSP and the s-EPSP were presynaptically inhibited by the 5- HT1D agonist sumatriptan. These data indicate that the s-EPSP is mediated by 5-HT acting upon 5-HT2 receptors. This represents strong support for the role of 5-HT as an excitatory neurotransmitter in the CNS. Further, it demonstrates that synaptic release of 5-HT can mediate opposing effects on the membrane potential of a single cell.