The general transcription factor TFIID is a large multisubunit complex required for the transcription of most protein-encoding genes by RNA polymerase II. Taking advantage of a TFIID preparation partially depleted in the initiator-binding Taf2p subunit, we determined the conformational and biochemical variations of the complex by electron tomography and cryo-electron microscopy of single molecules. Image analysis revealed the extent of conformational flexibility of the complex and the selection of the most homogeneous TFIID subpopulation allowed us to determine an improved structural model at 23 Angstroms resolution. This study also identified two subpopulations of Taf2p-containing and Taf2p-depleted TFIID molecules. By comparing these two TFIID species we could infer the position of Taf2p, which was confirmed by immunolabeling using a subunit-specific antibody. Mapping the position of this crucial subunit in the vicinity of Taf1p and of TBP sheds new light on its role in promoter recognition.