Alpha-adrenergic receptor, subtype 2A (alpha2A-AR), activation is one of the primary modes of action for norepinephrine (NE) in the rat hippocampal formation. In this study, alpha2A-AR immunoreactivity (alpha2A-AR-I) was localized by light and electron microscopy in the rat hippocampus and dentate gyrus by using a previously characterized antibody to the rat alpha2A-AR. By light microscopy, dense alpha2A-AR-I was observed in the pyramidal and granule cell layers. Diffuse and slightly granular alpha2A-AR-I was found in the neuropil in all other laminae, notably stratum lacunosum-moleculare. Ultrastructurally, alpha2A-AR-I was found in neuronal cytoplasm associated with large multivesicular-like organelles and with clusters adjacent to endoplasmic reticula and/or plasmalemma. The distribution of alpha2A-AR-I in the strata oriens, radiatum, and lacunosum-moleculare of hippocampal CA1 and CA3 regions and in the molecular layer of the dentate gyrus was remarkably similar (n > 2,000 profiles examined): alpha2A-AR-I was found in axons and terminals (approximately 40%), glia (approximately 30%), dendritic spines (approximately 25%), and dendritic shafts (approximately 5%). This mixed pre- and postsynaptic distribution was not seen in the stratum lucidum of the CA3 region and the dentate hilar region, where most alpha2A-AR-I was found in axons (approximately 60%) and glia (approximately 30%). Alpha-2A-AR-labeled axons were small and unmyelinated; labeled terminals usually formed asymmetric synapses on unlabeled spines; and labeled dendritic spines were morphologically similar to pyramidal or granule cells. Dual labeling studies demonstrated that some axons contained alpha2A-AR-I and tyrosine hydroxylase (TH), the catecholaminergic synthesizing enzyme, and that some TH-labeled terminals were in close proximity to alpha2A-AR-labeled spines and glia. These studies demonstrate that hippocampal alpha2A-AR-I is localized (1) presynaptically in both noncatecholaminergic and catecholaminergic terminals, (2) postsynaptically in the dendritic spines of pyramidal and granule cells near catecholaminergic terminals, and (3) in some glial processes. These results suggest several sites for NE to exert its effects on hippocampal alpha2A-ARs.