Here we describe the establishment and prediction utilities for a novel nine-amino-acid transactivation domain, 9aa TAD, that is common to the transactivation domains of a large number of yeast and animal transcription factors. We show that the 9aa TAD motif is required for the function of the transactivation domain of Gal4 and the related transcription factors Oaf1 and Pip2. The 9aa TAD possesses an autonomous transactivation activity in yeast and mammalian cells. Using sequence alignment and experimental data we derived a pattern that can be used for 9aa TAD prediction. The pattern allows the identification of 9aa TAD in other Gal4 family members or unrelated yeast, animal, and viral transcription factors. Thus, the 9aa TAD represents the smallest known denominator for a broad range of transcription factors. The wide occurrence of the 9aa TAD suggests that this domain mediates conserved interactions with general transcriptional cofactors. A computational search for the 9aa TAD is available online from National EMBnet-Node Austria at http://www.at.embnet.org/toolbox/9aatad/.