A yeast TBP mutant (N2-1) is described here that is defective specifically in responding to acidic activators in vivo. N2-1 does not support activation by Gal4, Ace1, and Gcn4, but appears unaffected for constitutive transcription, repression by the Cyc8-Tup1 and Not complexes, and transcription by polymerase I (Pol) and Pol III. In vitro, N2-1 fails to interact with TFIIA, but it associates normally with a TATA element, an acidic activation domain, and TFIIB. Fusion of the small subunit of TFIIA to N2-1 restores activation function in vivo. Thus, an efficient interaction between TBP and TFIIA is required for transcriptional activation in vivo.