The duplex DNA unwinding ability of Xenopus transcription factor A was examined by treatment of plasmid-protein complexes with calf thymus topoisomerase I and resolution of the topoisomer distributions by agarose gel electrophoresis. Factor A binding to plasmids does result in a small degree of unwinding as evidenced by a shift in topoisomer distributions upon relaxation. Slightly more unwinding by factor A was observed on a specific plasmid, containing four tandemly repeated 5S RNA genes, when compared to a non-specific plasmid of nearly identical size containing no 5S specific sequences. The amount of unwinding observed on the specific plasmid, about one base-pair per gene, suggests that unwinding of the DNA duplex is not an important aspect of binding of factor A to the 5S RNA gene.