Abstract Activation of neurons in the rostral ventral medulla, by electrical stimulation or microinjection of glutamate, produces antinociception. Microinjection of opioid compounds in this region also has an antinociceptive effect, indicating that opioids activate a medullary output neuron that exerts a net inhibitory effect on nociception. When given systemically in doses sufficient to produce antinocieeption, morphine produces distinct, opposing responses in two physiologically identifiable classes of rostral medullary neurons. “Off-cells” are activated, and have been proposed to inhibit nociceptive transmission. “On-cells” are invariably depressed, and may have a pro-nociceptive role. Although on-cell firing is also depressed by iontophoretically applied morphine, off-cells do not respond to morphine applied in this manner. The present study used local infusion of the μ-selective opioid peptide Tyr- d-Ala-Gly-MePhe-Gly-ol-enkephalin (DAMGO) within the rostral medulla to determine whether off-cells are activated by an opioid action within this region that is sufficient to produce a behaviorally measurable antinociception. Activity of on- and off-cells was recorded before and after local infusion of DAMGO noxious heat-evoked tail flick reflex was inhibited in 17 of 28 cases. On-cell firing was profoundly depressed, and this occurred irrespective of the antinociceptive effectiveness of the injection. Off-cells were activated following DAMGO microinjections, but only in experiments in which the tail flick reflex was inhibited. Both reflex inhibition and neuronal effects were reversed following systemic administration of naloxone. These observations thus confirm the role of the on-cell as the focus of direct opioid action within the rostral medulla, and strongly support the proposal that disinhibition of off-cells is central to the antinociception actions of opioids within this region.