Multiple chromatographically separable complexes containing the TATA binding protein (TBP), which exhibit different functional properties, exist in HeLa cells. At least three distinct subpopulations of such complexes can be functionally defined as TFIID since they function with RNA polymerase II. Using a partially reconstituted HeLa cell in vitro transcription system and immunoprecipitation with a monoclonal antibody directed against TBP, we show that stimulation of transcription by the chimeric activators GAL-VP16, GAL-TEF-1 and GAL-ER(EF) requires the presence of factors which are tightly associated with these TFIID complexes. Moreover, the activity of GAL-TEF-1 appears to be mediated by at least two chromatographically distinct populations of TFIID. The factor(s) associated with one of these populations is also required for the activity of GAL-ER (EF) and GAL-VP16, while the factor(s) associated with the other population functions selectively with GAL-TEF-1. These two TFIID populations are composed of both common and unique TBP associated factors (TAFs).