1. Whole-cell recordings were made from seventy-seven identified rat sympathetic preganglionic neurones (SPN) in spinal cord slices. Perfusion of 5-HT (0.5-30 microM) strongly depolarized 90% of neurones. The response was slow in onset, could last over 10 min and was associated with an increase in input resistance. 5-HT could also evoke rhythmical membrane potential oscillations in a population of previously quiescent neurones. 2. The 5-HT response persisted in TTX and also in low-Ca(2+)-high-Mg2+ artificial cerebrospinal fluid (ACSF), suggesting that the receptors are on SPN. The 5-HT uptake inhibitor 6-nitroquipazine potentiated the 5-HT-induced depolarization. 3. The 5-HT-induced depolarization was reduced and then abolished by membrane hyperpolarization to potentials of about -100 mV, but was not reversed in sign by further hyperpolarization. In voltage clamp, 5-HT evoked inward currents associated with the reduction of an outwardly rectifying potassium conductance. 4. The 5-HT2 receptor agonist alpha-methyl-5-HT mimicked the 5-HT response on all neurones, as did the 5-HT1 receptor agonist 5-carboxamidotryptamine (5-CT) on 71% of SPN. The responses to 5-HT, alpha-methyl-5-HT and 5-CT were inhibited by the 5-HT2 antagonists ketanserin and ritanserin. 5. Pressure ejection of 5-HT over the central canal region could evoke a biphasic inhibitory-excitatory response. This response persisted in TTX, suggesting that an inhibitory 5-HT receptor may be located on the medial dendrites. 6. SPN are powerfully depolarized by 5-HT acting at 5-HT2 receptors, via the closure of an outwardly rectifying potassium conductance. The long duration of the response and the ability of 5-HT to induce rhythmical oscillations suggest that 5-HT may have an important role in regulating SPN excitability.