The teleost Mauthner (M)-cell is subjected to a powerful glycinergic inhibition that regulates its threshold for initiation of a vital escape reflex. The effects of 5-HT on this inhibition were studied in current- and voltage-clamp experiments because the M-cell and its afferents have a profuse serotonergic innervation, including axoaxonal contacts with terminals of the inhibitory interneurons. Local applications of the amine and/or of its uptake blockers markedly enhanced inhibitory currents evoked by synchronous activation of two identified presynaptic networks. In order to determine the site of this 5-HT action, we exploited the fact that in the M-cell, synaptic noise is predominantly inhibitory. Furthermore, its quantal components can be resolved providing a means to distinguish pre- and post-synaptic loci underlying variations in synaptic efficacy. As in other cell types, it consists of spontaneous exocytotic events and multiquantal responses triggered by action potentials in afferent neurons. 5-HT or its uptake blockers produced a long-lasting enhancement of this noise, manifested as a shift to the right of amplitude distribution histograms of the individual inhibitory postsynaptic currents (IPSCs) and an increase in the mean quantal content of noise. In contrast, the size of the miniature IPSC (mIPSCs) remained constant in these experiments and in another series where 5-HT was given in presence of TTX. In this case, the amine also increased the rate of occurrence of single quanta. Taken together, these data indicate that 5-HT acts presynaptically to increase the probability of evoked and spontaneous release of glycine. This conclusion was reinforced by evidence that the overall frequency of the responses in noise could be unaltered by 5-HT, indicating that the afferent firing pattern had remained constant. Extracellular recordings of the presynaptic volleys showed that action potentials were not prolonged and suggested that 5-HT closes K+ channels in the terminal membrane, leading to an increase of resistance and a larger depolarization in that region as a possible mechanism for the facilitation of release. While the enhancement of transmitter release persisted for 20 min or more, there was also a transient induction of a K+ inward-rectifying current in the M-cell. These results indicate that 5-HT increases the efficacy of inhibitory synaptic transmission in the vertebrate CNS by a presynaptic action.