The homeobox-containing gene, Emx1, a mouse homologue of Drosophila empty spiracles, is specifically expressed in the developing telencephalic cortex. It has been reported that Emx1 transcripts and the protein product are localized in most cells of the cerebral cortex during the process of proliferation, migration, differentiation and maturation. We provide evidence here, based on a multitude of experimental approaches in developing rats, in support of the hypothesis that the expression of this gene is restricted to pyramidal neurons. Specifically, we found that, similar to pyramidal neurons, cells expressing Emx1 are distributed in all cortical layers, except layer I. Using in situ hybridization and immunocytochemistry at the light and electron microscope levels, we have shown that the density, distribution, soma shape and ultrastructural features of these cells were identical to those of pyramidal neurons. Double-labelling experiments confirmed that the vast majority of Emx1-expressing cells also contained glutamate, a marker of pyramidal neurons. We also found that this gene is expressed by most glutamate-containing neurons in dissociated cortical cell cultures and the vast majority of cells in radially arranged clones of pyramidal cells in the cortices of chimeric mice. Thus, the homeobox gene Emx1 can be reliably used as a marker of the pyramidal cell lineage.