Mammalian TFIIIB can be separated into two fractions required for transcription of the adenovirus type 2 VAI gene, which have been designated 0.38M-TFIIIB and 0.48M-TFIIIB. While 0.48M-TFIIIB has not been characterized, 0.38M-TFIIIB corresponds to a TBP-containing complex. We describe here the purification of this complex, which consists of TBP and a closely associated polypeptide of 88 kDa, and the isolation of a cDNA corresponding to the 88-kDa polypeptide. The predicted protein sequence reveals that the 88-kDa polypeptide corresponds to a human homolog of the Saccharomyces cerevisiae BRF protein, a subunit of yeast TFIIIB. Human BRF (hBRF) probably corresponds to TFIIIB90, a protein previously cloned by Wang and Roeder (Proc. Natl. Acad. Sci. USA 92:7026-7030, 1995), although its predicted amino acid sequence differs from that reported for TFIIIB90 over a stretch of 67 amino acids as a result of frameshifts. Immunodepletion of more than 90 to 95% of the hBRF present in a transcription extract severely debilitates transcription from the tRNA-type VAI promoter but does not affect transcription from the TATA box-containing human U6 promoter, suggesting that the 0.38M-TFIIIB complex, and perhaps hBRF as well, is not required for U6 transcription.