The Drosophila homeotic selector (HOM) genes encode a family of DNA binding transcription factors that specify developmental fates of different body segments by differentially regulating the activity of downstream target genes. A central question is how the HOM proteins achieve their developmental specificity despite the very similar DNA binding specificities of isolated HOM proteins in vitro. Specificity could be achieved by differential interactions with protein cofactors. The extradenticle gene might encode such a cofactor since it interacts genetically in parallel with Ultrabithorax, abdominal-A, and perhaps other HOM genes. By using a yeast two-hybrid system, we demonstrate selective interaction of the extradenticle homeodomain protein with certain Ultrabithorax and abdominal-A proteins but not with an Antennapedia protein or a more distant homeodomain protein. Strong interaction with Ultrabithorax proteins requires only the Ultrabithorax homeodomain and a 15-residue N-terminal extension that includes Tyr-Pro-Trp-Met (YPWM), a tetrapeptide motif found near the homeodomain in most HOM proteins and their mammalian Hox counterparts. The size and sequence of the region between the YPWM element and the homeodomain differ among Ultrabithorax isoforms, and this variable region appears to affect the interaction detected in the assay.