Immunocytochemical studies have shown that nuclear and extranuclear estrogen receptors (ERs) are present in several extra-hypothalamic brain regions. The goal of this study was to determine the subcellular location of functional ERs, particularly extranuclear ERs, by demonstrating ¹²⁵I-estradiol binding in the rat forebrain and medullary sections prepared for light and electron microscopic autoradiography. Some sections were immunocytochemically labeled with the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), prior to the autoradiographic procedure. By light microscopy, dense accumulations of silver grains denoting ¹²⁵I-estradiol binding were observed over cells in the ventromedial and arcuate hypothalamic nuclei, the amygdala and the nucleus of the solitary tract (NTS). In sections labeled for TH, large accumulations of silver grains were admixed with TH-labeled processes in the medial nucleus of the amygdala, and over TH-labeled perikarya in the medial and commissural NTS. Electron microscopic analyses were focused on the rostral ventrolateral medulla (RVLM) and the hippocampal CA1 region, two regions previously shown to have extranuclear ERs. In the RVLM, silver grains indicative of ¹²⁵I-estradiol binding were found within a few large terminals, affiliated with mitochondria. In the hippocampus, autoradiographic silver grains denoting ¹²⁵I-estradiol binding were associated with mitochondria in dendritic shafts or were near synaptic specializations on dendritic spines. These patterns of silver grain labeling were not seen in sections from rats that received ¹²⁵I-estradiol combined with "cold" estradiol. The association of ¹²⁵I-estradiol binding with pre- and postsynaptic profiles supports a functional role for non-nuclear ERs in brain.