Homeodomain proteins are believed to direct developmental pathways during Drosophila embryogenesis by the specific regulation of other genes. An unresolved issue is whether it is the homeodomain or the other regions of such proteins that confer target specificity. To test the role of the homeodomain in determining target specificity, we replaced the homeobox of Deformed with the homeobox of Ultrabithorax. The resulting chimeric protein cannot activate transcription from the Deformed gene, as does the normal Deformed protein. Instead, the chimeric protein activates ectopic transcription of Antennapedia, a gene normally regulated by Ultrabithorax. Our results indicate that in the context of the developing embryo, even closely related homeodomain sequences have different target specificities.