The analgesic actions of opioids can be modified by endogenous “anti-opioid” peptides, among them cholecystokinin (CCK). CCK is now thought to have a broader, pronociceptive role, and contributes to hyperalgesia in inflammatory and neuropathic pain states. The aim of this study was to determine whether anti-opioid and pronociceptive actions of CCK have a common underlying mechanism. We showed previously that a low dose of CCK microinjected into the rostral ventromedial medulla (RVM) blocked the analgesic effect of systemically administered morphine by preventing activation of off-cells, which are the antinociceptive output of this well characterized pain-modulating region. At this anti-opioid dose, CCK had no effect on the spontaneous activity of these neurons or on the activity of on-cells (hypothesized to facilitate nociception) or “neutral cells” (which have no known role in pain modulation). In this study, we used microinjection of a higher dose of CCK into the RVM to test whether activation of on-cells could explain the pronociceptive action of this peptide. Paw withdrawal latencies to noxious heat and the activity of a characterized RVM neuron were recorded in rats lightly anesthetized with methohexital. CCK (30 ng/200 nl) activated on-cells selectively and produced behavioral hyperalgesia. Firing of off-cells and neutral cells was unaffected. These data show that direct, selective activation of RVM on-cells by CCK is sufficient to produce thermal hyperalgesia and indicate that the anti-opioid and pronociceptive effects of this peptide are mediated by actions on different RVM cell classes.