Segmental identities along the insect body depend on the activities of Hox genes [1,2]. In Drosophila melanogaster, one well-studied Hox regulatory target is Distal-less (Dll), which is required for the development of distal limb structures [3]. In abdominal segments, Dll transcription is prevented when Hox proteins of the Bithorax Complex (BX-C) bind to cis-regulatory elements upstream of the Dll transcription start site [4,5]. Previous evolutionary comparisons of gene expression patterns suggest that this direct repression is conserved between Diptera and Lepidoptera, but is absent in the Crustacea [6,7]. We examined gene expression patterns in three orders of hexapods, all of which develop abdominal appendages, in order to determine when the strong repressive interaction between BX-C proteins and Dll appeared during evolution. In each of the species examined, Dll expression was initiated in abdominal cells despite the presence of high levels of BX-C proteins. It appears that the strong repressive effects of BX-C proteins on Dll expression arose relatively late in insect evolution. We suggest that the regulatory interaction between the BX-C genes and Dll has evolved within the hexapods in a complex, segment-specific manner.