Eukaryotic transcriptional activators function, at least in part, by promoting assembly of the preinitiation complex, which comprises RNA polymerase II and its general transcription factors (GTFs). Activator-mediated stimulation of the assembly of the preinitiation complex has been studied in vitro but has been relatively refractory to in vivo analysis. Here we use a DNA-crosslinking/immunoprecipitation assay to study in living cells the first step in the assembly of the preinitiation complex, the interaction between the TATA-box-binding protein (TBP) and its binding site, the TATA box. Analysis of a variety of endogenous yeast genes, and of a series of activators of differing strength, reveals a general correlation between TBP binding and transcriptional activity. Using mutant yeast strains, we show that Mot1 prevents the binding of TBP to inactive promoters and that activator-mediated stimulation of TBP binding requires additional GTFs, including TFIIB and Srb4. Taken together, our results indicate that TBP binding in vivo is stringently controlled, and that the ability of activators to stimulate this step in the assembly of the preinitiation complex is a highly cooperative process involving multiple transcription factors.