Opioid antinociception appears to be mediated at least in part by a pathway that projects from the periaqueductal gray (PAG) to the rostral ventromedial medulla (RVM), but the relationship between opioid receptors and PAG-RVM projection neurons is unclear. Previous electrophysiological studies have suggested that opioids act directly on some PAG neurons projecting to the RVM. However, immunoreactivity for neither the cloned mu-opioid receptor (MOR1) nor the cloned delta-opioid receptor (DOR1) has been observed in PAG cells retrogradely labeled from the RVM. In the present study, we examined the expression of DOR1 and MOR1 mRNAs in PAG neurons projecting to RVM using quantitative in situ hybridization and retrograde tract-tracing. Mesencephalic neurons were labeled in three male Sprague-Dawley rats by microinjection of Fluoro-Gold into the RVM. Five micrometer cryostat sections were cut and in situ hybridization was performed using full-length cRNA probes labeled with 35S-UTP. Retrogradely labeled neurons that were also labeled for MOR1 or DOR1 mRNA were observed in the dorsomedial, lateral, and ventrolateral portions of the PAG. Quantification was performed in the dorsomedial and ventrolateral PAG using the physical disector. We found that of 219 retrogradely labeled neurons, 50 +/- 14% expressed DOR1 mRNA. In a second set of 120 Fluoro-Gold-labeled neurons, 27 +/- 8% expressed MOR1 mRNA. Significantly more PAG-RVM projection neurons were labeled for MOR1 mRNA in the ventrolateral subregion of the PAG than in the dorsomedial subregion. However, no significant difference was observed in the proportions of retrogradely labeled neurons labeled for DOR1 mRNA in the ventrolateral subregion compared to the dorsomedial subregion. We conclude that opioids are likely to exert direct effects on PAG-RVM projection neurons through both delta- and mu-opioid receptors. In addition, direct effects on PAG-RVM projection neurons from activation of MOR1 appear more likely to be exerted in the ventrolateral PAG than in the dorsomedial PAG.