The Emx1 gene is a mouse homologue of a Drosophila head gap gene, empty spiracles. Corpus callosum defects have been reported for mutant mice lacking the Emx1 gene, indicating a possible involvement of the Emx1 gene in the regulation of axon guidance during development. However, it has recently come to light that genetic background could influence the outcome of the corpus callosum defects in mutant mice generated by ES cell technology. To investigate whether the corpus callosum defects resulted directly from Emx1 gene inactivation, we backcrossed the Emx1 mutant mice into the C57BL/6 background and found that the Emx1 homozygous mutant mice with the C57BL/6 background showed a normal corpus callosum score even though the cross-sectional area was 8% less than that of their wild-type littermates, indicating that genetic background could change the pattern of corpus callosum defects in Emx1 mutant mice. Furthermore, the indusium griseum and taenia tecta were always present and the cerebral cortical layers were well differentiated in Emx1 mutant mice. These results suggest that inactivation of the Emx1 gene does not contribute directly to the defects of corpus callosum and other brain structures associated with Emx1 mutant mice derived from a 129/Sv background.