Hox transcription factors, in combination with cofactors such as PBC proteins, provide diverse developmental fates to cells on the anteroposterior body axis of animal embryos. However, the mechanisms by which the different Hox proteins and their cofactors generate those diverse fates remain unclear. Recent findings have provided support for a model where the DNA binding sites that directly interact with Hox-PBC heterodimers determine which member of the Hox protein family occupies and thereby regulates a given target element. In the experiments reported here, we test the function of chimeric Hox response elements and, surprisingly, find evidence that runs counter to this view. A 21 bp cofactor binding sequence from an embryonic Deformed Hox response element, containing no Hox or Hox-PBC binding sites, was combined with single or multimeric sites that bind heterodimers of Labial-type Hox and PBC proteins. Normally, multimerized Labial-PBC binding sites are sufficient to trigger a Labial-specific activation response in either Drosophila or mouse embryos. Here we find that the 21 bp sequence element plays an important role in Deformed specificity, as it is capable of switching a Labial-PBC binding site/response element to a Deformed response element. Thus, cofactor binding sites that are separate and distinct from homeodomain binding sites can dictate the regulatory specificity of a Hox response element.