5-Hydroxytryptamine (5-HT) neurons in the midbrain dorsal raphe nucleus (DRN) are implicated in the drug treatment and pathophysiology of a wide variety of neuropsychiatric disorders. Accumulating evidence suggests that 5-HT6 receptors may be located and functional in the DRN; therefore, 5-HT6 receptor ligands may have potential as novel modulators of 5-HT neurotransmission. The current study investigated the effect of intravenous (i.v.) administration of the selective 5-HT6 receptor agonist, WAY-181187, and antagonist, SB-399885, on the firing of 5-HT neurons in the DRN in vivo. Extracellular recordings were made in the DRN of anesthetized rats, and single 5-HT neurons were identified on the basis of electrophysiological properties combined with juxtacellular labeling and postmortem immunohistochemical analysis. WAY-181187 (1-4 mg/kg i.v.) caused a dose-dependent increase in 5-HT neuron firing rate. In comparison, SB-399885 (0.125-1 mg/kg i.v.) caused a dose-dependent decrease in 5-HT neuron firing rate, an effect reversed by WAY-181187 (3 mg/kg i.v.). These effects of WAY-181187 and SB-399885 were observed in two separate sets of experiments. In summary, the current data show the modulation of 5-HT neuronal firing by the 5-HT6 ligands WAY-181187 and SB-399885 and are consistent with the presence of 5-HT6 receptor-mediated positive feedback control of 5-HT neurons.