Limited information indicates that the regulatory factor X1 (RFX1), the prototype member of the transcription factor RFX family, may play a role in the central nervous system. Our recent study showed that knockout of the Rfx1 gene led to early embryonic death. In the present study, we showed that heterozygous Rfx1(+/-) mice were fertile and grew normally. An abundant amount of RFX1 proteins were expressed in the olfactory bulb, hippocampus and cerebral cortex as detected by β-galactosidase staining (the gene knockout vector contained a coding region for β-galactosidase) and immunofluorescent staining with an anti-RFX1 antibody. RFX1 positive immunostaining was mainly in the nuclei of neurons and microglial cells and was absent from the astrocytes of mouse brains. The heterozygous Rfx1(+/-) mice expressed RFX1 mRNA and proteins at a level similar to that in the wild-type mice in the olfactory bulb and hippocampus. The expression level of RFX1 proteins was similar in the brains of mice ranging from 15 day old embryos to four month old adults. Our results suggest a significant expression of RFX1 proteins in the mammalian brain. This expression is cell-type and brain-region specific and may take a random monoallelic expression pattern.