Characterization of their transcription activation domains is critical to understanding functional specificity within the Hox family of proteins. However, few Hox activation domains have been identified and none characterized in detail. In this study, promotor-reporter assays in yeast and Drosophila S2 cell culture were used to refine the boundaries of the activation domain of the Drosophila Hox protein Ultrabithorax (Ubx) and to identify critical elements within this domain. We found that residues 159-242 were sufficient for 50% function, and full transactivation capacity was achieved with inclusion of additional N-terminal sequences. Activation domain sequence and placement relative to the homeodomain differ between Ubx and other Hox proteins, consistent with the possibility that diverse activation mechanisms contribute to functional distinctions in vivo. The essential residues 159-242 in the UbxIb activation domain are predicted to contain a beta-sheet segment followed by an alpha-helix. This putative alpha-helical region was established to be necessary, but not sufficient, for transcriptional activation. Disruption of the helix by proline substitutions abolished activation function, while alteration of side chains presented on the surface of this putative helix with alanine or lysine mutations had no significant effect on activity. Collectively, these data indicate that this secondary structural element is a key component in forming an effective activation domain in the UbxIb protein. Interestingly, the alpha-helix critical for transcriptional activation is found only for Ubx orthologs from flies and not other species. The mutant Ubx proteins generated in this study have potential applications in deciphering Hox functions in vivo.