Insects bear a stereotyped set of limbs, or ventral body appendages. In the highly derived dipteran Drosophila melanogaster, the homeodomain transcription factor encoded by the Distal-less (Dll) gene plays a major role in establishing distal limb structures. We have isolated the DEI orthologue (TcDll) from the beetle Tribolium castaneum, which, unlike Drosophila, develops well-formed limbs during embryogenesis, TcDll is initially expressed at the sites of limb primordia formation in the young embryo and subsequently in the distal region of developing legs, antennae and mouthparts except the mandibles. Mutations in the Short antennae (Sa) gene of Tribolium delete distal limb structures, closely resembling the Dll phenotype in Drosophila, TcDll expression is severely reduced or absent in strong So alleles, Genetic mapping and molecular analysis of Sa alleles also support the conclusion that TcDll corresponds to the So gene. Our data indicate functional conservation of the Dll gene in evolutionarily distant insect species. Implications for evolutionary changes in limb development are discussed.