The rostral portion of the ventral medial medulla (RVM) is a crucial site for the supraspinal antinociceptive actions of opioids. Previous studies have reported that serotonergic antagonists block the analgesia induced by microinjection of morphine into the RVM (Hammond and Yaksh [1984] Brain Res 298:329-337) and that spinally projecting serotonergic RVM neurons express mu-opioid receptors (MOR) (Kalyuzhny et al. [1996] J Neurosci 16:6490-6503; Wang and Wessendorf [1999] J Comp Neurol 404:183-196). In addition, axons immunoreactive for the endogenous MOR ligand endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) (EM-2) have been reported to be in the RVM (Martin-Schild et al. [1999] J Comp Neurol 405:450-471; Pierce and Wessendorf [2000] J Chem Neuroanat 18:181-207). In the present study we examined the relationship of EM-2-immunoreactive (EM-2-ir) axons to serotonergic and nonserotonergic RVM neurons in rats, including neurons projecting to the dorsal spinal cord. We also examined the origins of EM-2-ir in the RVM. Using unbiased methods we estimated the total number of cells in the RVM to be 1.50 x 10(4) and of these up to 70% were retrogradely labeled from the dorsal spinal cord. EM-2-ir fibers apposed both serotonergic and nonserotonergic RVM neuronal profiles. However, serotonergic profiles were significantly more likely to be apposed than nonserotonergic profiles. Thus, although serotonergic neurons comprise a minority of RVM neurons (23% of the total RVM neurons), they appear to be selectively apposed by EM-2-ir fibers. We also found that hypothalamic EM-2-ir neurons, but not EM-2-ir neurons, in the nucleus of the solitary tract projected their axons to the RVM.