Abstract: Mediator is one of the most important co-activators that function in eukaryotic transcriptional regulation. In Saccharomyces cerevisiae, Mediator is comprised of 25 subunits belonging to four structurally distinct modules: Head, Middle, Tail, and Cyc-C. Although each module plays a critical role in the regulation of a distinct set of genes, the precise molecular mechanisms remain unclear. To gain new insight into the role of the less-characterized Middle module, we analyzed the function of Med9 by constructing a set of mutants and subjecting them to a range of in vivo and in vitro assays. Our results demonstrate that Med9 has two functional domains. The species-specific amino-terminal half (aa 1-63) plays a regulatory role in transcriptional regulation in vivo and in vitro. In contrast, the well-conserved carboxy-terminal half (aa 64-149) has a more fundamental function involved in direct binding to the amino-terminal portions of Med4 and Med7 and the assembly of Med9 into the Middle module. Importantly, activator-dependent recruitment of TBP and Taf11 to the promoter is differentially affected in med9 extracts and in extracts lacking Mediator. Add-back experiments indicate that some unidentified factor(s) in med9 extracts may impact the binding of TFIID to the promoter.