Regulated transcription of class II genes of the yeast Saccharomyces cerevisiae requires the diverse functions of mediator complex. In particular, MED6 is essential for activated transcription from many class II promoters, suggesting that it functions as a key player in the relay of activator signals to the basal transcription machinery. To identify the functional relationship between MED6 and other transcriptional regulators, we conducted a genetic screen to isolate a suppressor of a temperature-sensitive (ts) med6 mutation. We identified an SRB4 allele as a dominant and allele-specific suppressor of med6-ts. A single missense mutation in SRB4 can specifically suppress transcriptional defects caused by the med6 ts mutation, indicating a functional interaction between these two mediator subunits in the activation of transcription. Biochemical analysis of mediator subassembly revealed that mediator can be dissociated into two tightly associated subcomplexes. The Med6 and Srb4 proteins are contained in the same subcomplex together with other dominant Srb proteins, consistent with their functional relationship revealed by the genetic study. Our results suggest not only the existence of a specific interaction between Med6 and Srb4 but also the requirement of this interaction in transcriptional regulation of RNA polymerase II holoenzyme.