We have analyzed transcription factor-mediated DNA supercoiling catalyzed by the Xenopus oocyte extract (S-150). Under conditions that inhibit endogenous supercoiling activity (2 mM EDTA), the 5S RNA specific transcription factor, TFIIIA, promotes a negative change in DNA linking number. The SV40 binding protein, T antigen, appears not to promote DNA supercoiling under these conditions. A nucleosomal ladder can be seen after DNase I digestions only if the DNA template is pre-bound by TFIIIA prior to the addition of the S-150 extract. These studies suggest that TFIIIA may stimulate DNA supercoiling by enhancing the development of protein-DNA interactions via a mechanism that may include nucleosome assembly.