Activation of mu opioid receptors (MORs) has a net excitatory effect in the hippocampal formation through inhibition of gamma-amino butyric acid (GABA)-containing interneurons. To determine the precise subcellular targets of MOR agonists, immunoreactivity against MOR1 and GABA was examined in single sections of the hippocampal formation prepared for dual-labeling electron microscopy. In both the CA1 region of hippocampus and the dentate gyrus, MOR-like immunoreactivity (-li) was present in neuronal somata, dendrites, axons, and axon terminals, as well as a very few glial processes. Axon terminals with MOR-li formed symmetric synapses with principal cell dendrites and somata. Many MOR-labeled profiles of all types also contained GABA-li, and the vast majority possessed the ultrastructural characteristics of interneurons. Additionally, in the dentate gyrus a very small proportion of granule cell dendrites contained MOR-li. MOR-li, identified using immunogold-silver particles, was often affiliated with the extrasynaptic regions of neuronal plasma membranes, consistent with responsiveness to diffusing endogenous neuropeptide ligands. Semiquantitative analysis of the distribution of MOR-li revealed significantly more "presynaptic" (axons and terminals) than "postsynaptic" (somata and dendrites) labeled profiles in most laminae. We conclude that in addition to previously described somatodendritic MOR-li, a substantial amount of MOR-li in hippocampal formation is presynaptic. Furthermore, MORs are almost exclusively in GABAergic interneurons.