Vertebrate Hox genes display nested and overlapping patterns of expression. During mouse hindbrain development, Hoxb3 and Hoxb4 share an expression domain caudal to the boundary between rhombomeres 6 and 7. Transgenic analysis reveals that an enhancer (CR3) is shared between both genes and specifies this domain of overlap. Both the position of CR3 within the complex and its sequence are conserved from fish to mammals, suggesting it has a common role in regulating the vertebrate HoxB complex. CR3 mediates transcriptional activation by multiple Hox genes, including Hoxb4, Hoxd4, and Hoxb5 but not Hoxb1. It also functions as a selective HOX response element in Drosophila, where activation depends on Deformed, Sex combs reduced, and Antennapedia but not labial. Taken together, these data show that a Deformed/Hoxb4 autoregulatory loop has been conserved between mouse and Drosophila. In addition, these studies reveal the existence of positive cross-regulation and enhancer sharing as two mechanisms for reinforcing the overlapping expression domains of vertebrate Hox genes. In contrast, Drosophila Hox genes do not appear to share enhancers and where they overlap in expression, negative cross-regulatory interactions are observed. Therefore, despite many well documented aspects of Hox structural and functional conservation, there are mechanistic differences in Hox complex regulation between arthropods and vertebrates.