1. The effects of microiontophoretic applications of acetylcholine (ACh), (-)-noradrenaline ((-)-NA) and 5-hydroxytryptamine (5-HT) have been investigated on spontaneously active brain stem neurones in decerebrate unanaesthetized rats and in rats anaesthetized with either tribromoethanol, urethane or pentobarbitone.2. Four types of responses to both (-)-NA and 5-HT were seen. These were: simple excitation; excitation preceded by a short-lasting inhibition; short-lasting inhibition and prolonged inhibition. Three types of responses to ACh were seen: an excitation with long latency of onset; excitation with short latency of onset, resembling the response to an excitant amino acid, and a short-lasting inhibitory response.3. The types of responses to microiontophoretically applied ACh, (-)-NA or 5-HT in anaesthetized and unanaesthetized animals were similar.4. The number of ACh excitatory responses with short latency of onset were significantly reduced in the pentobarbitone-anaesthetized group and a small but significant increase in the number of 5-HT inhibitory effects were observed in each anaesthetized group of animals.5. A significantly greater proportion of slower firing neurones (less than 10 spikes/s) were found in the pentobarbitone-anaesthetized animals.6. The effects of microiontophoretically applied and i.v. administered pentobarbitone were studied on spontaneously active neurones which responded consistently to ACh and a control agonist.7. Pentobarbitone administered by either route reduced the firing rate of most neurones studied and was shown to antagonize specifically the excitation of neurones by exogenously applied ACh.8. It is suggested that postsynaptic antagonism of endogenously released ACh may be a contributing factor in the mechanism of action of pentobarbitone.