1. Current and voltage recordings were made from antidromically identified motoneurones (MNs) in transverse thoracolumbar spinal cord slices of neonatal rats. 2. Applied by superfusion (10-100 microM) or pressure ejection, 5-hydroxytryptamine (5-HT) elicited a slow depolarization (or inward current) in 81% and a hyperpolarization (or outward current) in 9% of responsive MNs; the responses persisted in a low-Ca2+, high-Mg2+ or tetrodotoxin (TTX)-containing solution. 3. 5-HT induced the occurrence in some MNs of excitatory postsynaptic potentials (EPSPs) or inhibitory postsynaptic potentials (IPSPs), which were reversibly eliminated by TTX, low-Ca2+, high-Mg2+ solution or by the 5-HT2 receptor antagonists ketanserin and spiperone. Also, kynurenic acid and strychnine abolished, respectively, the 5-HT-induced EPSPs and IPSPs. 4. The 5-HT depolarization was associated with increased membrane resistance, was reduced by hyperpolarization and nullified near -100 mV. The extrapolated reversal potential was shifted to a positive direction in elevated [K+]o. 5. The depolarizing response was mimicked by the 5-HT2 receptor agonist (+2-)-1(2,5-dimethyoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) and blocked by 5-HT antagonists methysergide and cyproheptadine and by 5-HT2 antagonists ketanserin and spiperone; methiothepin and MDL 72222 were without effect. 6. The 5-HT hyperpolarization was associated with decreased membrane resistance. The 5-HT1A agonist 8-hydroxy-2-(di-N-propylamino) tetralin hydrobromide (8-OH-DPAT) mimicked the hyperpolarizing response. 7. Single or repetitive (10-30 Hz) electrical stimuli elicited in about 30% of MNs, in addition to a fast EPSP, a slow EPSP with electrophysiological characteristics similar to that of 5-HT induced depolarization. Methysergide and spiperone abolished the slow EPSPs evoked in some of these MNs. 8. It is suggested that 5-HT, acting on 5-HT2 and 5-HT1A receptors, depolarizes and hyperpolarizes the MNs by decreasing and increasing K+ conductance. Additionally, 5-HT activates, via 5-HT2 receptors, excitatory and inhibitory interneurones, thereby indirectly affecting the activity of MNs. More importantly, 5-HT released from intraspinal nerves appears to be the mediator of a slow EPSP in a population of MNs.