The Brx1 homeobox gene has been isolated and shown to be expressed in the zona limitans intrathalamica (ZLI) of the mouse embryo. Brx1 is a member of the Brx gene family and comprises the genes for Brx1a and Brx1b, which differ in the sequence in the region located on the 5'-terminal side of the homeobox. The complete amino acid sequences of the open reading frame of Brx1a and Brx1b were determined and each was found to be similar to that of Rgs, the mouse homologue of the Rieger syndrome associated human RIEG gene (RGS), to the extent that the sequence of Rgs has been clarified. Brx1 was strongly expressed in the mammillary area as well as in the ZLI of the mouse embryonic brain. Homologues of Brx1a and Brx1b were isolated in chick in which the expression of Brx1 in the ventral diencephalon was well conserved. The expression of Brx1 along with that of Sonic hedgehog (Shh), Nkx2.2, Dlx1 and Arx was examined at the time of the formation of ZLI in mouse embryos. The expression of Shh was initially noted in the ventricular zone of the presumptive ZLI and was then replaced by that of Brx1 at the time of radial migration of the neuroepithelial cells. Nkx2.2 was widely expressed in the ventricular zone of presumptive ZLI and also as a narrow band in the mantle zone. The expression of Dlx1 and Arx in the presumptive ventral thalamus extended as far as ZLI and overlapped with that of Brx1. The Dlx1- and Arx-expressing cells in ZLI, which extended towards the lateral (pial) surface of the diencephalic wall, differed from those expressing Nkx2.2 and Brx1. The embryonic ventral lateral geniculate nucleus present in the visual pathway was eventually formed from these cells. Each homeobox gene was also expressed regionally in the nucleus, suggesting that the nucleus is comprised of subdivisions.