Multiple stages of protein-DNA interaction in the assembly of RNA polymerase III transcription complexes on a Saccharomyces cerevisiae 5S rRNA gene have been distinguished by DNase I "footprinting" and gel retardation. Transcription factor IIIA interacts with approximately 35 base pairs of the internal promoter region. Transcription factors IIIC and IIIB incrementally extend the interaction along the 5S gene, if, and only if, transcription factor IIIA is also bound. Complexes assembled from the complete set of purified transcription factors or from a complete transcription system extend over the entire transcription unit together with almost 50 base pairs of 5' flanking sequence.