Relaxation of Xenopus 5S plasmid DNA (pX1o8) in the presence of transcription factor (TF) IIIA reduces the linking number of the DNA. Parallel experiments with plasmid pMB9 or cloned hepatitis B viral DNA indicate a degree of non-specific unwinding by TF; however, 60% of the effect observed for pX1o8 is due to specific interaction of TF IIIA with the 5S rRNA gene internal promoter sequence. The extent of unwinding (0.2-0.4 helical turn per TF IIIA binding site) is not consistent with the complete denaturation of the 50-base-pair TF binding site; however, it is consistent with a change in helix rotation, denaturation of 2-4 nucleotides per binding site, or DNA wrapping about a protein core. We show that proteins other than TF IIIA (bovine serum albumin and RNase) have no effect on the linking number of DNA when present during relaxation and that the unwinding activity associated with TF is heat labile. These results suggest that TF IIIA may facilitate transcription by altering the helical configuration of 5S DNA.