ABSTRACT Emx 1 and 2 are the murine homologues of the Drosophila empty spiracles gene and based on their expression pattern may be involved in the regional specification of the mammalian forebrain. During early embryogenesis, Emx2 is expressed in the presumptive cerebral cortex and olfactory bulbs and later, in the hippocampus proper and dentate gyrus. The latter are involved in memory processes. To understand the role of Emx2 in vivo, we have mutated the gene in mice. Homozygous embryos die postnatally because of severe urogenital alterations. These mice present cerebral hemispheres with a reduced size and exhibit specific morphological alterations in allocortical structures of the medial wall of the brain. The dentate gyrus is missing and the hippocampus proper is reduced. The medial limbic cortex is also severely shortened. The development of the dentate gyrus is affected at the onset of its formation with defects in the neuroepithelium from which it originates. These findings demonstrate that Emx2 is required for the development of several forebrain structures.