RNA polymerase I and II transcription factors SL1 and TFIID, respectively, are composed of the TATA-binding protein (TBP) and a set of TBP-associated factors (TAFs) responsible for promoter recognition. How the universal transcription factor TBP becomes committed to a TFIID or SL1 complex has not been known. Complementary DNAs encoding each of the three TAF(I)s that are integral components of SL1 have now been isolated. Analysis of subunit interactions indicated that the three TAF(I)s can bind individually and specifically to TBP. In addition, these TAF(I)s interact with each other to form a stable TBP-TAF complex. When TBP was bound first by either TAF(I)110, 63, or 48, subunits of TFIID such as TAF(II)250 and 150 did not bind TBP. Conversely, if TBP first formed a complex with TAF(II)250 or 150, the subunits of SL1 did not bind TBP. These results suggest that a mutually exclusive binding specificity for TBP intrinsic to SL1 and TFIID subunits directs the formation of promoter- and RNA polymerase-selective TBP-TAF complexes.