TFIIIB, the central transcription initiation factor of the eukaryotic nuclear RNA polymerase (pol) III is composed of three subunits: the TATA-binding protein; Brf, the TFIIB-related subunit; and BX, the Saccharomyces cerevisiae, TFC5 gene product. The orientation of the BX subunit within the TFIIIB-DNA complex has been analyzed at two promoters by two approaches that involve site-specific photochemical protein-DNA cross-linking: a collection of BX internal and external deletion proteins has been surveyed for those deletions that alter the interaction of BX with DNA or change the orientation of BX relative to DNA; a method for regionally mapping cross-links between specific DNA sites and (32)P-end-labeled protein has also been applied. The results map an N-proximal segment of BX to the upstream end of the TFIIIB-DNA complex and amino acids 299-315 to the principal DNA-contact site, approximately 8 base pairs upstream of the TATA box. The analysis also indicates that a segment comprising amino acids 316-434 loops away from DNA, and locates the C-proximal 170 amino acids of BX downstream of the TATA box. Examination of two-cross-link products formed by DNA with adjacent and nearby photoactive nucleotides supports the conclusion that Brf and BX share an extended interface along the length of the TFIIIB-DNA complex.