RBP is a cellular protein that functions as a transcriptional repressor in mammalian cells. RBP has elicited great interest lately because of its established roles in regulating gene expression, inDrosophila and mouse development, and as a component of the Notch signal transduction pathway. This report focuses on the mechanism by which RBP represses transcription and thereby regulates expression of a relatively simple, but natural, promoter. The results show that, irrespective of the close proximity between RBP and other transcription factors bound to the promoter, RBP does not occlude binding by these other transcription factors. Instead, RBP interacts with two transcriptional coactivators: dTAF_II110, a subunit of TFIID, and TFIIA to repress transcription. The domain of dTAF_II110 targeted by RBP is the same domain that interacts with TFIIA, but is disparate from the domain that interacts with Sp1. Repression can be thwarted when stable transcription preinitiation complexes are formed before RBP addition, suggesting that RBP interaction with TFIIA and TFIID perturbs optimal interactions between these coactivators. Consistent with this, interaction between RBP and TFIIA precludes interaction with dTAF_II110. This is the first report of a repressor specifically targeting these two coactivators to subvert activated transcription.