The dorsal raphe (DR) nucleus is a system of nuclei lying in the midline of the lower brainstem constituting the largest collection of serotonin-containing neurons in the brain. The DR nucleus, which is under the influence of various synaptic inputs containing the excitatory amino acids (EAAs), serotonin and noradrenaline (NA), has been reported to be involved in the process of nociception. We studied the effects of fentanyl and morphine on the firing activity of neurons within the DR nucleus in the rat brain slice preparation in vitro using extracellular recording techniques. The decreased activity of DR neurons induced by these opioids was reversed upon application of naloxone. Since almost all neurons were silent, N-methyl-D-aspartate (NMDA) and noradrenaline were used to excite the DR neurons. Serotonin contained in the DR nucleus acts as a neuromodulator to enhance the excitatory effects of NMDA. All DR neurons were excited by NMDA, whereas 72% of the neurons were excited by NA. NA has a long-lasting effect on the firing activity of the neurons, although the firing pattern was less regular than that induced by NMDA. Of the total number of the DR neurons excited by NMDA and/or NA, 71% were inhibited by fentanyl and 73% by morphine. The results support our hypothesis that these opioids inhibit the firing activity of DR neurons and, in turn, might alter nociception directly and/or indirectly by modifying the central serotonergic system.