A novel mouse homeobox-containing gene, Nkx-2.2, has been isolated. Nkx-2.2 is a member of a family of genes whose homeodomains are homologous to that of the Drosophila NK-2 gene. Nkx-2.2 transcripts are found in localized domains of the brain during mouse embryogenesis. Nkx-2.2 expression in the brain abuts and partially overlaps with the expression domains of two other related homeobox-containing genes, TTF-1 and Dlx. The expression domains of the three genes in the developing prosencephalon coincide with anatomical boundaries, particularly apparent in the diencephalon. This result raises the possibility that these genes may specify regional differentiation of the developing diencephalon into its anatomically and functionally defined subregions. Nkx-2.2 may be involved in specifying diencephalic neuromeric boundaries.