Microinjection of kappa opioid receptor (KOR) agonists into the rostral ventromedial medulla (RVM) attenuates mu-opioid receptor mediated antinociception and stress-induced analgesia, yet is also reported to have an analgesic effect. To determine how KOR agonists produce both antinociceptive and anti-analgesic actions within the RVM, the KOR agonist U69593 was microinjected directly into the RVM while concurrently monitoring tail flick latencies and RVM neuronal activity. Among RVM neurons recorded in vivo, two types show robust changes in activity just prior to the nocifensive tail flick reflex: on-cells burst just prior to a tail flick and their activity is pro-nociceptive, whereas off-cells pause just prior to the tail flick and their activity is antinociceptive. Although RVM microinjection of U69593 did not affect tail flick latencies on its own, it did attenuate the on-cell burst, an effect blocked by co-injection of the KOR antagonist, nor-binaltorphimine (nor-BNI). Furthermore, U69593 inhibited ongoing activity in subsets of off-cells (4/11) and neutral cells (3/9). Microinjection of U69593 into the RVM also attenuated morphine antinociception and suppressed the excitation of off-cells. Together with previous in vivo and in vitro studies, the present results are consistent with the idea that KOR agonists can be either pro-nociceptive through direct inhibition of off-cells, or anti-analgesic through both postsynaptic inhibition and presynaptic inhibition of glutamate inputs to RVM off-cells.