We have isolated a novel homeobox gene that is expressed in the vertebrate central nervous system and which shows striking similarity to the Drosophila al gene in the homeodomain (85% identity) and in a 17 amino acid-sequence near the carboxyl-terminus. This gene was designated Arx (aristaless related homeobox gene) in consideration of its structural similarity to the al gene. Arx was highly conserved between mouse and zebrafish. Neuromeric expression in the forebrain and longitudinal expression in the floor plate were observed in mouse and zebrafish. The expression of Arx in the ganglionic eminence and ventral thalamus overlapped regionally with that of Dlx1, but the cell layer where Arx is expressed differed from that of the Dlx1. This gene was also found to be expressed in the dorsal telencephalon (presumptive cerebral cortex) of mouse embryos. The structure and expression pattern of Arx with respect to any possible relationship to al and Dlx1, as well as the function of Arx in the floor plate are discussed.