Pax9 belongs to the Pax family of transcriptional regulators that are defined by a highly conserved DNA-binding region, the paired domain. Drosophila, mouse and human genetics have shown that Pax proteins play multiple roles in tissue patterning and organogenesis by mediating their functions in a highly tissue-specific manner. Members of the Pax family, Pax9 and Pax1, act synergistically during vertebral formation. However, only Pax9 is essential for tooth formation. Furthermore, mutations of PAX9 are associated with human tooth agenesis. The highly tooth-specific molecular functions of Pax9 suggest that its activity is tightly regulated. Most likely, this occurs through interactions with other protein factors. Among the regulatory molecules that are expressed in dental mesenchyme, the Msx1 homeoprotein is of particular interest. The closely overlapping expression patterns of Pax9 and Msx1 are consistent with a role in epithelial-mesenchymal interactions. To demonstrate that Pax9 interacts with Msx1 physiologically in vivo and in vitro, we performed co-immunoprecipitation and GST interaction assays. Our results indicate that there is a physical association between the two proteins. Our biochemical data, coupled with human genetic studies and expression analysis in a mouse model, indicate a functional relationship between Pax9 and Msx1 during tooth development.