Six4 is a member of the Six family genes, homologues of Drosophila melanogaster sine oculis. The gene is thought to be involved in neurogenesis, myogenesis, and development of other organs, based on its specific expression in certain neuronal cells of the developing embryo and in adult skeletal muscles. To elucidate the biological roles of Six4, we generatedSix4-deficient mice by replacing the Sixhomologous region and homeobox by the β-galactosidase gene. 5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside staining of the heterozygous mutant embryos revealed expression ofSix4 in cranial and dorsal root ganglia, somites, otic and nasal placodes, branchial arches, Rathke's pouch, apical ectodermal ridges of limb buds, and mesonephros. The expression pattern was similar to that of Six1 except at the early stage of embryonic day 8.5. Six4-deficient mice were born according to the Mendelian rule with normal gross appearance and were fertile. No hearing defects were detected. Six4-deficient embryos showed no morphological abnormalities, and the expression patterns of several molecular markers, eg, myogenin andNeuroD3 (neurogenin1), were normal. Our results indicate that Six4 is not essential for mouse embryogenesis and suggest that other members of the Six family seem to compensate for the loss of Six4.