It has been generally accepted that the TATA binding protein (TBP) is a universal mediator of transcription by RNA polymerase I, II, and III. Here we report that the TBP-related factor TRF1 rather than TBP is responsible for RNA polymerase III transcription in Drosophila. Immunoprecipitation and in vitro transcription assays using immunodepleted extracts supplemented with recombinant proteins reveals that a TRF1:BRF complex is required to reconstitute transcription of tRNA, 5S and U6 RNA genes. In vivo, the majority of TRF1 is complexed with BRF and these two proteins colocalize at many polytene chromosome sites containing RNA pol III genes. These data suggest that in Drosophila, TRF1 rather than TBP forms a complex with BRF that plays a major role in RNA pol III transcription.