In the ventrolateral periaqueductal gray (PAG), endogenous pathways which dampen pain transmission can be activated by either opioids or excitatory amino acids such as N-methyl D-aspartate (NMDA). The effects of these ligands may converge, because morphine-produced analgesia in the PAG can be blocked by NMDA receptor antagonists. To determine the relationship between the subcellular sites where opioid ligands of the mu opioid receptor (MOR) and NMDA receptor ligands may act, we studied the ultrastructural distribution of immunolabeling for MOR and the R1 subunit of the NMDA receptor (NR1) in the ventrolateral PAG. MOR labeling was most commonly distributed along extrasynaptic regions of the plasma membrane of neuronal dendrites (80% or 245/306). In addition, MOR labeling was found presynaptically in axon terminals (13% or 39/306) which preferentially formed symmetric (inhibitory-type) synapses. NR1 immunoreactivity was also prevalent in dendrites (72% or 242/335), but in contrast to MOR, was usually associated with a subset of postsynaptic densities. Axon terminals (5%, 17/335) and glial processes (18%, 61/335) comprised the remainder of NR1-labeled profiles. There was a striking colocalization of MOR and NR1 labeling within dendrites. The majority of NR1-labeled dendrites contained MOR labeling (72%, 176/242) and likewise, the majority of MOR-labeled dendrites contained NR1 labeling (72%, 176/245). Thus, mu opioid and NMDA receptor ligands may act at several overlapping subcellular sites to modulate behaviors subserved by the ventrolateral PAG, such as antinociception.