In vitro transcription of templates containing deletion-substitution mutations has localized two essential promoter elements of the 5 S rRNA gene of Saccharomyces cerevisiae. A promoter element spanning the start site of transcription extends from -14 to +8, and a short internal control region (ICR) extends from +81 to +94. Changes in spacing between these elements by more than a few base pairs significantly reduce transcription. The site of RNA polymerase III transcription factor A (TFIIIA) binding, mapped by determination of the G residues that are protected from methylation on exposure of the TFIIIA.5 S DNA complex to dimethyl sulfate, is coincident with the ICR. Incorporation of TFIIIC into the TFIIIA.5 S rRNA gene complex protects additional G residues 5' and 3' of the ICR from methylation.