Estrogen has been shown to play a modulatory role in cerebellar neuronal signaling. Recent reports have also shown that estrogen receptor beta (ER-beta) mRNA is expressed in cerebellum. The purpose of the present study was to identify and map ER-beta protein expression, and to determine the identity of the major splice variants of ER-beta mRNA in the cerebellum. Polyclonal antibodies raised against the NH(3)- and COOH-termini of rat ER-beta were used for immunohistochemistry. Mapping of ER-beta immunoreactivity was compared with the distribution of ER-beta mRNA using in situ hybridization. We also determined, using RT-PCR, whether the ER-beta mRNA was variably spliced in cerebellum. Our results show that in all cases the distribution of ER-beta protein was identical to the distribution of ER-beta mRNA. Both Purkinje cells and scattered cells in the granule cell layer, perhaps golgi cells, robustly expressed ER-beta. Reverse transcription-polymerase chain reaction analysis showed that three splice variants in addition to wild type ER-beta are expressed in rat cerebellum. However, wild type ER-beta was the predominant form. These observations provide anatomical evidence that neurons in the cerebellum express ER-beta and thus may be targets of estrogen action.