Homeodomain proteins are transcriptional regulatory factors that, in general, bind DNA with relatively low sequence specificity and affinity. One mechanism homeodomain proteins use to increase their biological specificity is through interactions with other DNA-binding proteins. We have examined how the yeast (Saccharomyces cerevisiae) homeodomain protein alpha2 specifically interacts with Mcm1, a MADS box protein, to bind DNA specifically and repress transcription. A patch of predominantly hydrophobic residues within a region preceding the homeodomain of alpha2 has been identified that specifies direct interaction with Mcm1 in the absence of DNA. This hydrophobic patch is required for cooperative DNA binding with Mcm1 in vitro and for transcriptional repression in vivo. We have also found that a conserved motif, termed YPWM, frequently found in homeodomain proteins of insects and mammals, partially functions in place of the patch in alpha2 to interact with Mcm1. These findings suggest that homeodomain proteins from diverse organisms may use analogous interaction motifs to associate with other proteins to achieve high levels of DNA binding affinity and specificity.