TFC5, the unique and essential gene encoding the BX component of the Saccharomyces cerevisiae RNA polymerase III transcription factor (TF)IIIB has been cloned. It encodes a 594-amino acid protein (67,688 Da). Escherichia coli-produced BX has been used to reconstitute entirely recombinant TFIIIB that is fully functional for TFIIIC-directed, as well as TATA box-dependent, DNA binding and transcription. The DNase I footprints of entirely recombinant TFIIIB, composed of BX, the 67-kDa Brf, and TATA box-binding protein, and TFIIIB reconstituted with natural BX are indistinguishable. A truncated form of BX lacking 39 N-terminal and 107 C-terminal amino acids is also functional for transcription.