Hesx1 is a highly conserved homeobox gene present in vertebrates, but absent from invertebrates. Gene targeting experiments in mice have shown that this transcriptional repressor is required for normal forebrain and pituitary development. In humans, mutations in HESX1 impairing either its repressing activity or DNA binding properties lead to a comparable phenotype to that observed in Hesx1 deficient mice. In an attempt to gain insights into the molecular function of HESX1, we have performed a yeast two-hybrid screen and identified DNA methyltransferase 1 (DNMT1) as a HESX1 binding protein. We show that Dnmt1 is co-expressed with Hesx1 within the anterior forebrain and in the developing Rathke's pouch. Mapping of the interacting regions indicates that the entire HESX1 protein is required to establish binding to a portion of the N-terminus of DNMT1 and its catalytic domain in the C-terminus. The HESX1-DNMT1 complexes can be immunoprecipitated in cells and co-localise in the nucleus. These results establish a link between HESX1 and DNMT1 and suggest a novel mechanism for the repressing properties of HESX1.