Cholinergic septohippocampal neurons are affected by circulating estrogens. Previously, we found that extranuclear estrogen receptor-α (ERα) immunoreactivity in presynaptic profiles had an overlapping distribution with cholinergic afferents in the rat hippocampal formation. To determine the subcellular relationships between cholinergic presynaptic profiles and ERα, hippocampal sections were dually immunolabeled for vesicular acetylcholine transporter (VAChT) and ERα and examined by electron microscopy. Within the hippocampal formation, immunoreactivities for VAChT and ERα both were presynaptic, although their subcellular targeting was distinct. VAChT immunoreactivity was found exclusively within presynaptic profiles and was associated with small synaptic vesicles, which usually filled axon terminals. VAChT-labeled presynaptic profiles were most concentrated in stratum oriens of the hippocampal CA1 region and dentate inner molecular layer and hilus. In contrast, ERα immunoreactivity was found in clusters affiliated either with select vesicles or with the plasmalemma within preterminal axons and axon terminals. ERα-immunoreactive (IR) presynaptic profiles were more evenly distributed between hippocampal lamina than VAChT-IR profiles. Quantitative ultrastructural analysis revealed that VAChT-IR presynaptic profiles contained ERα immunoreactivity (ranging from 3% to 17%, depending on the lamina). Additionally, VAChT-IR presynaptic profiles apposed ERα-IR dendritic spines, presynaptic profiles, and glial profiles; many of the latter two types of profiles abutted unlabeled dendritic spines that received asymmetric (excitatory-type) synapses from unlabeled terminals. The presence of ERα immunoreactivity in cholinergic terminals suggests that estrogen could rapidly and directly affect the local release and/or uptake of acetylcholine. The affiliation of cholinergic terminals with excitatory terminals near ERα-labeled dendritic spines or glial profiles suggests that alterations in acetylcholine release could indirectly affect estrogen-modulated structural plasticity. J. Comp. Neurol. 463:390–401, 2003. © 2003 Wiley-Liss, Inc.