Estrogen receptor alpha (ER alpha) and ER beta are members of the steroid nuclear receptor family that modulate gene transcription in an estrogen-dependent manner. ER mRNA and protein have been detected both peripherally and in the central nervous system, with most data having come from the rat. Here we report the development of an ER beta-selective antibody that cross-reacts with mouse, rat, and human ER beta protein and its use to determine the distribution of ER beta in the murine brain. Further, a previously characterized polyclonal antibody to ER alpha was used to compare the distribution of the two receptors in the first comprehensive description of ER distribution specifically in the mouse brain. ER beta immunoreactivity (ir) was primarily localized to cell nuclei within select regions of the brain, including the olfactory bulb, cerebral cortex, septum, preoptic area, bed nucleus of the stria terminalis, amygdala, paraventricular hypothalamic nucleus, thalamus, ventral tegmental area, substantia nigra, dorsal raphe, locus coeruleus, and cerebellum. Extranuclear-ir was detected in several areas, including fibers of the olfactory bulb, CA3 stratum lucidum, and CA1 stratum radiatum of the hippocampus and cerebellum. Although both receptors were generally expressed in a similar distribution through the brain, nuclear ER alpha-ir was the predominant subtype in the hippocampus, preoptic area, and most of the hypothalamus, whereas it was sparse or absent from the cerebral cortex and cerebellum. Collectively, these findings demonstrate the region-selective expression of ER beta and ER alpha in the adult ovariectomized mouse brain. These data provide an anatomical framework for understanding the mechanisms by which estrogen regulates specific neural systems in the mouse.