SummaryThe modular Mediator complex is a coactivator of RNA polymerase II transcription. We show that depletion of the main complex scaffold Med14 or the head module scaffold Med17 is lethal and results in global transcriptional downregulation in yeast, though Med17 removal has a markedly greater negative effect. Depletion of Med14 or Med17 impairs pre-initiation complex (PIC) assembly similarly, suggesting that the differential transcriptional effects observed are not due to differing extents of defective PIC formation. Co-depletion of Med14 and Med17 reduced transcription and TFIIB promoter occupancy similarly to Med17 ablation alone, suggesting that the independent head module can weakly stimulate transcriptionin vivo, though not to a level that maintains viability. We suggest that, while the structural integrity of complete Mediator and the head module are both important for PIC assembly, the head module additionally promotes optimal PIC function and is thus the key functional module of Mediator in this regard.