Estrogens regulate neural processes such as neuronal development, reproductive behavior, and hormone secretion, and signal through estrogen receptor (ER) alpha and ERbeta (here called ERbeta1). Recent studies have found variations in ERalpha and ERbeta1 mRNA splicing in rodents and humans. Functional reporter gene assays suggest that these splicing variations alter ER-mediated transcriptional regulation. Estrogen receptor beta 2 (ERbeta2), an ERbeta1 splice variant containing an 18 amino acid (AA) insert in the ligand binding domain, binds estradiol with approximately 10-fold lower affinity than ERbeta1, suggesting that it may serve as a low-affinity ER. Moreover, ERbeta2 reportedly acts in a dominant-negative fashion when heterodimerized with ERbeta1 or ERalpha. To explore the function of ERbeta2 in brain, an antiserum (TwobetaER.1) targeting the 18 AA insert was developed and characterized. Western blot analysis and transient expression of ERbeta2 in cell lines demonstrated that TwobetaER.1 recognizes ERbeta2. In the adult female rat brain, ERbeta2 immunoreactivity is localized in the cell nucleus and is expressed with a distribution similar to that of ERbeta1 mRNA. ERbeta2 immunoreactive cell numbers were high in, for example, piriform cortex, paraventricular nucleus, supraoptic nucleus, arcuate nucleus, and hippocampal CA regions, whereas it was low in the dentate gyrus. Moreover, ERbeta2 is coexpressed in gonadotropin-releasing hormone and oxytocin neurons. These studies demonstrate ERbeta splice variant proteins in brain and support the hypothesis that ER signaling diversity depends not only on ligand or coregulatory proteins, but also on regional and phenotypic selectivity of ER splice variant proteins.