The factor(s) responsible for the adenovirus E1A-stimulated transcription of RNA polymerase III genes was localized previously in a chromatographic fraction containing transcription factor IIIC (TFIIIC). In further studies, two distinct forms of TFIIIC, which were chromatographically separable, generated VA gene-protein complexes that were distinguished by gel shift assays. The form of TFIIIC that generated the more slowly migrating promoter complex had greater transcriptional activity in vitro, associated more rapidly with the promoter, and formed a more salt-resistant complex. Greater amounts of this more active form of TFIIIC resulted from either E1A expression during infection or growth of the cells in a higher concentration of serum, whereas template commitment assays indicated that overall TFIIIC concentrations remained unchanged during viral infection. The in vitro interconversion of the two forms of TFIIIC by phosphatase treatment suggests that transcriptional activation of RNA polymerase III genes can be mediated by phosphorylation of TFIIIC.