Neurons in the rostral ventromedial medulla (RVM) are important in the opioid modulation of dorsal horn nociceptive transmission. Systemically administered morphine inhibits one class of RVM cells, the on-cells; excites a second class of RVM cells, the off-cells; and has no effect on a third class, neutral cells. In contrast, iontophoretic application of morphine inhibits on-cells but does not alter the activity of either off- or neutral cells. The present study addresses whether the differential sensitivity to exogenous opioids is correlated with a differential termination pattern onto the three classes of RVM neurons by afferents containing endogenous opioids. Intracellular recordings were made from RVM neurons in rats under light halothane anesthesia. Physiologically characterized neurons were injected with Neurobiotin and then subsequently visualized with a Texas red fluorophore. Thick (50 microns) sections containing labeled RVM cells were processed for enkephalin immunoreactivity (ENK-IR) using an FITC fluorophore and then optically sectioned at 1.5 micron intervals using a dual-channel confocal laser scanning microscope. ENK-IR appositions were found on the somata and dendrites of all on-cells. Although ENK-IR varicosities were also apparently apposed to off- and neutral cells, the density of such appositions was significantly less than the density of ENK-IR appositions onto on-cells. The greater overall density of ENK-IR appositions onto on-cells was apparently due to a concentration of appositions on the soma and proximal dendrites of these neurons. These results support a model of RVM function in which endogenous opioid peptides produce an antinociceptive action by a direct inhibitory action on on-cells that facilitate nociceptive transmission. This on- cell inhibition may produce an additional antinociceptive effect by removing a possible on-cell inhibition of off-cells, which are thought to inhibit nociceptive transmission.