Serotonin modulates a variety of neural processes, and is present in a subpopulation of neurons in the raphe nuclei. To study their electrophysiological properties, cells from the mesopontine raphe nuclei of the neonatal rat were dissociated and grown for up to 10 weeks in microcultures. Approximately one third of the neurons were identified as serotonergic based on the presence of serotonin immunoreactivity, tryptophan hydroxylase immunoreactivity, or a high affinity monoamine transporter. About 5% of cultured raphe neurons contained tyrosine hydroxylase immunoreactivity, while 25% contained GABA immunoreactivity. However, no neurons contained both serotonin and tyrosine hydroxylase staining, and less than 1% displayed both serotonin and GABA immunoreactivities. Cultured serotonergic neurons did not exhibit pacemaker firing in the presence of alpha 1 adrenergic receptor agonists such as phenylephrine or norepinephrine. Approximately one third were hyperpolarized by serotonin or the selective serotonin1A receptor agonist, (+/-)-8-hydroxy-2-(di-N-propylamino)tetralin. Virtually all serotonergic neurons responded to application of glutamate, kainate, N-methyl-D-aspartate, GABA, and glycine. Depolarizing and hyperpolarizing synaptic potentials blocked by glutamate or GABAA receptor antagonists were frequently observed in both serotonergic and non-serotonergic raphe neurons. Slow inhibitory postsynaptic potentials were evoked by activating single presynaptic serotonergic neurons with a brief intracellular current pulse. The slow inhibitory synaptic potential had a mean latency to onset of 35 +/- 5 ms, a duration of 0.8-2.6 s, and was inhibited by the serotonin1A autoreceptor antagonists, (-)propranolol and spiperone. The rising and falling phases of the inhibitory potential could be fit by single exponential functions with mean time constants of 53 +/- 8 ms and 504 +/- 78 ms, respectively. Serotonin1A receptor-mediated autoinhibition was observed in microcultures containing a solitary serotonergic neuron, and thus constituted synaptic serotonin release, responsiveness, and re-uptake by a single vertebrate neuron. In summary, histochemical and electrophysiological evidence was obtained for catecholaminergic, GABAergic, and glutamatergic non-serotonergic raphe neurons in culture, many of which formed functional synaptic connections with neighboring cells. Additionally, cultured mesopontine serotonergic neurons expressed many of the cytochemical markers, neurotransmitter receptors, and synaptic functions observed in such cells in vivo, but the proportion of neurons sensitive to serotonergic and adrenergic agonists was significantly less than that reported in vivo. For the first time, the kinetics and pharmacology of serotonergic synaptic transmission by a single vertebrate serotonergic raphe neuron were determined, and found to resemble those observed after extracellular stimulation of populations of raphe neurons in slices and in vivo.