mu-Opioid agonists frequently activate output neurons in the brain via disinhibition, that is, by inhibiting "secondary cells," which results in disinhibition of "primary cells," considered to be output neurons. Secondary cells are generally presumed to be inhibitory interneurons that serve only to regulate the activity of the output neurons. However, studies of the opioid-sensitive neurons in the rostral ventromedial medulla, a region with a well-documented role in nociceptive modulation, indicate that the opioid-inhibited neurons in this region (termed "on-cells" when recorded in vivo) have a distinct functional role that parallels and opposes the output of the subset of RVM neurons that are activated following opioid administration, the "off-cells." The aim of the present study was to analyze the relative timing of on- and off-cell reflex-related firing in the rostral ventromedial medulla to help determine whether on-cells are likely to function as inhibitory interneurons in this region. On- and off-cells display complementary firing patterns during noxious-evoked withdrawal: off-cells stop firing and on-cells show a burst of activity. If on-cells are inhibitory interneurons mediating the off-cell pause, the on-cells would be expected to begin their reflex-related discharge before the off-cells cease firing. To examine this we recorded activity of on- and off-cell pairs during heat-evoked paw or tail withdrawal in lightly anesthetized rats. For each cell pair, we measured the onsets of the off-cell pause and the on-cell burst. Contrary to what would be expected if on-cells were inhibitory interneurons, off-cells typically ceased firing before on-cells began reflex-related firing, with a mean 481 (+/-69) ms lag between the final off-cell spike and the first on-cell spike. This suggests that on-cells do not mediate the off-cell pause, and points instead to presynaptic mechanisms in opioid-mediated disinhibition of medullary output neurons. These data also support an independent role for on-cells in pain modulation.