Ascidians and vertebrates belong to the Phylum Chordata and both have dorsal tubular central nervous systems. The structure of the ascidian neural tube is extremely simple, containing less than 400 cells, among which less than 100 cells are neurons. Recent studies suggest that, despite its simple organization, the mechanisms patterning the ascidian neural tube are similar to those of the more complex vertebrate brain. Identification of homologous regions between vertebrate and ascidian nervous systems, however, remains to be resolved. Here we report the expression of HrPax-258 gene: an ascidian homologue of vertebrate Pax-2, Pax-5 and Pax-8 genes. Molecular phylogenetic analyses indicate that HrPax-258 is descendant from a single precursor gene that gave rise to the three vertebrate genes. The expression pattern of HrPax-258 suggests that this subfamily of Pax genes has conserved roles in regional specification of the brain. Comparison with expression of ascidian Otx (Hroth) and a Hox gene (HrHox1) by double-staining in situ hybridizations indicate that the ascidian brain region can be subdivided into three regions; the anterior region marked by Hroth probably homologous to the vertebrate forebrain and midbrain, the middle region marked by HrPax-258 probably homologous to the vertebrate anterior hindbrain (and maybe also midbrain) and the posterior region marked by Hox genes which is homologous to the vertebrate hindbrain and spinal cord. Later expression of HrPax-258 in atrial primordia implies that basal chordates such as ascidians have already acquired a sensory organ that develops from epidermal thickenings (placodes) and expresses HrPax-258; we suggest it is homologous to the vertebrate ear. Therefore, placodes are not likely to be a newly acquired feature in vertebrates, but may have already been possessed by the earliest chordates.