The yeast Gal4 protein, like many activators, binds TATA-binding protein (TBP) directly in vitro. It has been speculated that this protein-protein interaction is important for Gal4p-mediated activation of transcription, but little work has been done to test specific models involving this interaction. In this study, the effect of Gal4p on TBP-TATA binding is addressed. Specifically, it is asked if the Gal4p-TBP interaction can support cooperative binding of the two factors to promoters. It is easy to see how such an event could stimulate transcription, particularly from promoters with a non-consensus TATA box. In vitro, however, a derivative of Gal4p (Gal4-(1-93+768-881)) containing the DNA-binding, dimerization, and activation domains does not bind to promoter DNA cooperatively with either recombinant, purified TBP, or with protein from a yeast crude extract. In vivo, reporter gene experiments using promoters with differing TBP affinities reveal no major Gal4p-mediated stimulation of TBP function from weak TATA boxes, as would be predicted if the proteins bind cooperatively. Furthermore, native Gal4p and a potent Gal4p-based artificial activator lacking a TBP-binding activation domain support similar ratios of transcription from a series of promoters identical except for mutations in the TATA box. It is concluded that Gal4p and TBP do not bind cooperatively to promoters and that this mechanism does not contribute substantially to Gal4p-mediated transcriptional activation.