SUMMARY1. 5-Hydroxytryptamine (5-HT) and various 5-HT antagonists have been applied micro-electrophoretically from multibarrelled micropipettes into the environment of single neurones in the post-sigmoid and suprasylvian gyri of the cat cerebral cortex.2. In unanaesthetized animals (encephale isole) a high proportion of neurones (30 %) were excited by 5-HT. This excitation usually had a rapid onset and was seen both in spontaneously active neurones and in otherwise quiescent neurones in which firing was induced by L-glutamate. Some neurones were so sensitive that the uncontrolled diffusion from micropipettes was sufficient to excite them. More cells were excited by 5-HT applied as a cation from solutions of the bimaleate salt than when solutions of the creatinine sulphate salt were used.3. In a high proportion of cells (33 %) spontaneous firing or amino acid excitation was depressed by 5-HT. 4. A mixed effect was seen in a small proportion (6 %) of the cells tested; usually 5-HT caused an excitation initially which was followed by a depression. In other cells, desensitization occurred, and the excitatory effect of 5-HT was diminished or lost. In two cells, however, the depression was reversibly prevented by these antagonists.8. Some cells tested with 5-HT were also tested with ACh or (-noradrenaline. The response of a cell to ACh was not significantly related to its response to 5-HT. The degree of correlation between the responses to noradrenaline and 5-HT was large, but not statistically significant with the small number of cells studied. 9. The effects of 5-HT on cells in animals anaesthetized with oc-chloralose did not differ significantly from its effects in unanaesthetized preparations. It is suggested that the use of this anaesthetic may prove a useful alternative to unanaesthetized preparations.10. The systemic injection of small quantities of thiopentone sodium selectively and reversibly reduced the sensitivity of some units to excitation by 5-HT at a time when the response to glutamate was unaffected. On other occasions, the 5-HT excitation was unaffected, though the response to glutamate was reduced.11. These results are discussed in relation to the possible nature of the 5-HT receptors in the cerebral cortex, and the interfering effects of anaesthesia on the response of brain cells to potential transmitter substances.