Human ribosomal RNA synthesis by RNA polymerase I requires the activator UBF and the promoter selectivity factor SL1, which consists of the TATA binding protein (TBP) and three associated subunits, TAF(I) 110, TAF(I)63, and TAF(I)48. Here it is shown that both TAF(I)110 and TAF(I)63 contact the promoter, whereas TAF(l)48 serves as a target for interaction with UBF and is required to form a transcriptionally active SL1 complex responsive to UBF in vitro. TAF(I)48 also alters the ability of TBP to interact with TATA box elements, and the resulting complex fails to support transcription by RNA polymerase II. Thus, TAF(I)48 may function both as a target to mediate UBF activation and as a class-specific promoter selectivity factor.