The rostral ventromedial medulla (RVM) has recently received considerable attention in efforts to understand mechanisms of hyperalgesia and persistent pain states. Three classes of neurons can be identified in the RVM based on responses associated with nocifensive reflexes: on-cells, off-cells and neutral cells. There is now direct evidence that on-cells exert a net facilitating effect on spinal nociception, and that off-cells depress nociception. The present experiments tested whether the secondary hyperalgesia produced by topical application of mustard oil involves an activation of on-cells in RVM. Firing of a characterized RVM neuron and the latencies of withdrawal reflexes evoked by noxious heat were recorded in lightly anesthetized rats before and after application of mustard oil to the shaved skin of the leg above the knee. Mineral oil was applied as a control. Mustard oil produced a significant increase in ongoing and reflex-related discharge of on-cells, as well as a decrease in the activity of off-cells. Neutral cell firing was uniformly unchanged following application of mustard oil. The alterations in on- and off-cell firing were associated with a significant decrease in the latency to withdraw the paw of the treated limb from the heat stimulus, and this hyperalgesia was blocked by microinjection of lidocaine within the RVM. Withdrawals evoked by heating the contralateral hindpaw, forepaw and tail were unchanged following mustard oil application. These experiments support a pro-nociceptive role for on-cells, and suggest that these neurons contribute to secondary hyperalgesia in inflammation.