A number of mammalian multiprotein complexes containing homologs of Saccharomyces cerevisiae Mediator subunits have been described recently. High-molecular-mass complexes (1 to 2 MDa) sharing several subunits but apparently differing in others include the TRAP/SMCC, NAT, DRIP, ARC, and human Mediator complexes. Smaller multiprotein complexes (approximately 500 to 700 kDa), including the murine Mediator, CRSP, and PC2, have also been described that contain subsets of subunits of the larger complexes. To evaluate whether these different multiprotein complexes exist in vivo in a single form or in multiple different forms, HeLa cell nuclear extract was directly resolved over a Superose 6 gel filtration column. Immunoblotting of column fractions using antisera specific for several Mediator subunits revealed one major size class of high-molecular-mass (approximately 2-MDa) complexes containing multiple mammalian Mediator subunits. No peak was apparent at approximately 500 to 700 kDa, indicating that either the smaller complexes reported are much less abundant than the higher-molecular-mass complexes or they are subcomplexes generated by dissociation of larger complexes during purification. Quantitative immunoblotting indicated that there are about 3 x 10(5) to 6 x 10(5) molecules of hSur2 Mediator subunit per HeLa cell, i.e., the same order of magnitude as RNA polymerase II and general transcription factors. Immunoprecipitation of the approximately 2-MDa fraction with anti-Cdk8 antibody indicated that at least two classes of Mediator complexes occur, one containing CDK8 and cyclin C and one lacking this CDK-cyclin pair. The approximately 2-MDa complexes stimulated activated transcription in vitro, whereas a 150-kDa fraction containing a subset of Mediator subunits inhibited activated transcription.