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Saccharomyces cerevisiae Transcription Factor IIIA (TFIIIA) has been purified to apparent homogeneity. Two polypeptides copurified with TFIIIA activity. Yeast TFIIIA is a DNA-binding protein which exhibits a high affinity for the internal control region of the homologous 5 S ribosomal RNA gene. Characterization of the yeast protein indicates that it shares most, but not all, of the molecular properties of its Xenopus TFIIIA counterpart.