Systemic administration of a cannabinoid agonist produces antinociception through the activation of pain modulating neurons in the rostral ventromedial medulla (RVM). The aim of the present study was to determine how a cannabinoid receptor agonist acting directly within the RVM affects neuronal activity to produce behaviorally measurable antinociception. In lightly anesthetized rats, two types of RVM neurons have been defined based on changes in tail flick-related activity. On-cells increase firing (on-cell burst), whereas off-cells cease firing (off-cell pause), just prior to a tail flick. The cannabinoid receptor agonist WIN55,212-2 was microinfused directly into the RVM while monitoring tail flick latencies and on- and off-cell activity. Microinfusion of WIN55,212-2 (2.0 microg/microl and 0.4 microg/microl) reduced the tail flick-related on-cell burst, decreased the duration of the off-cell pause, and increased off-cell ongoing activity. These changes were prevented by co-infusing the CB1 receptor antagonist, SR141716A (0.35 microg/microl), with WIN55,212-2 (0.4 microg/microl). Furthermore, 2.0 microg/microl WIN55,212-2 delayed the onset of the off-cell pause and increased tail flick latencies. Microinfusion of WIN55,212-2 to brain regions caudal or lateral to the RVM had no effect on RVM neuronal activity or tail flick latencies. These results indicate that cannabinoids act directly within the RVM to affect off-cell activity, providing one mechanism by which cannabinoids produce antinociception.