In yeast, a single form of TFIIIB is required for transcription of all RNA polymerase III (pol III) genes. It consists of three subunits: the TATA box-binding protein (TBP), a TFIIB-related factor, BRF, and B′′. Human TFIIIB is not as well defined and human pol III promoters differ in their requirements for this activity. A human homolog of yeast BRF was shown to be required for transcription at the gene-internal 5S and VA1 promoters. Whether or not it was also involved in transcription from the gene-external human U6 promoter was unclear. We have isolated cDNAs encoding alternatively spliced forms of human BRF that can complex with TBP. Using immunopurified complexes containing the cloned hBRFs, we show that while hBRF1 functions at the 5S, VA1, 7SL and EBER2 promoters, a different variant, hBRF2, is required at the human U6 promoter. Thus, pol III utilizes different TFIIIB complexes at structurally distinct promoters.