The Mediator is a multiprotein transcriptional coactivator that is expressed ubiquitously in eukaryotes from yeast to mammals and is required for induction of RNA polymerase II (pol II) transcription by DNA binding transcription factors. In the work described here, we exploit multidimensional protein identification technology (MudPIT) to carry out a proteomic analysis of the subunit composition of the mammalian Mediator complex. By comparing MudPIT data sets obtained from six independent Mediator preparations immunoaffinity purified through their Nut2 (MED10), Med25 (MED9), Intersex (MED29), LCMR1 (MED19), AK007855 (MED28), or CRSP70 (MED26) subunits, we identify a set of consensus mammalian Mediator subunits. In addition, we identify as Mediator-associated proteins the CDK8-like cyclin-dependent kinase CDK11 and the TRAP240-like KIAA1025 protein (MED13L), which is mutated in patients with the congenital heart defect transposition of the great arteries (TGA).