Abstract: Biochemical evidence suggesting that the predominant form of Mediator in the yeast Saccharomyces cerevisiae might be one in which the complex is associated with RNA polymerase II to form a holoenzyme has led to proposition of a holoenzyme-based model for transcription initiation. We report that polymerasefree Mediator, isolated early on during a whole-cell extract fractionation protocol, is in fact the most abundant form of the Mediator complex. The existence of free Mediator would make possible independent recruitment of Mediator and RNA polymerase II to the preinitiation complex. This is in agreement with reports from in vivo studies of time and spatial independence of Mediator and RNA polymerase II promoter interaction, with current models of preinitiation complex structure in which promoter DNA upstream of the transcription start site is positioned between Mediator and polymerase, and with the proposed role of Mediator as the major component of the Scaffold complex involved in transcription reinitiation.