The potent C-terminal activation domain of the RelA (p65) subunit of the cellular transcription factor NF-kappa B is shown to contain several discrete acidic activation modules. These short, approximately 11-amino-acid modules were able to give rise to only a low level of transcription activation when fused to the GAL4 DNA-binding domain as monomers. However, dimers and higher-order multimers activated the transcription of minimal promoter elements as effectively as the full-length RelA or VP16 activation domain. Therefore, this 11-amino-acid RelA-derived acidic module appears to contain all of the sequence information required to fully activate a target promoter element as long as it is presented in a form that permits functional synergy. Critical primary sequence requirements for acidic activation module function included a core phenylalanine residue and flanking bulky hydrophobic residues. Overall negative charge was necessary but not sufficient for function. While dimeric forms of the 11-amino-acid acidic activation module bound to either TFIIB or TATA-binding protein efficiently in vitro, a similarly charged peptide lacking the core phenylalanine residue failed to interact. Overall, these data demonstrate that the biological activity of the RelA activation domain is dependent on acidic activator sequences that are closely comparable to those detected in the activation domain of the viral VP16 regulatory protein. We hypothesize that the ability of these acidic activators to specifically interact with multiple components of the transcription initiation complex likely underlies the dramatic functional synergy exhibited by this class of activation domains in vivo.