Abstract: Mot1 is an essential yeast Snf2/Swi2-related ATPase that exerts both positive and negative effects on gene expression. In vitro, Mot1 can disrupt TATA-binding protein-DNA complexes in an ATP-dependent reaction. This activity can explain Mot1-mediated transcriptional repression, but how Mot1 activates transcription is unknown. We demonstrate that, remarkably, Mot1 is localized in vivo to promoters for both Mot1-repressed and Mot1-activated genes. Moreover, Mot1 ATPase activity is required for both activation and repression of gene activity. These findings suggest a novel function for the Mot1 ATPase at activated genes, perhaps involving ATP-driven reorganization of the preinitiation complex. Mot1 regulates the expression of approximately 3% of yeast genes in cells grown in rich medium. Most of these genes are repressed by Mot1, consistent with Mot1's ATP-dependent TATA-binding protein-DNA dissociating activity. Additionally, approximately 77% of the Mot1-repressed genes are involved in the diauxic shift, stress response, mating, or sporulation. The gene sets controlled by NC2 and Srb10 are strongly correlated with the Mot1-controlled set, suggesting that these factors cooperate in transcriptional control on a global scale.