The CD spectrum of a restriction fragment that contains a single copy of a Xenopus borealis somatic 5 S rRNA gene, like those of two smaller fragments from the binding site for transcription factor IIIA (TFIIIA), is that of B-form DNA. Under dehydrating conditions (76% ethanol) the 5 S rDNA undergoes a transition into an A conformation. The spectra of the three fragments, however, do exhibit some perturbation in that the longwave positive peaks are shifted to shorter wavelengths and have enhanced rotational strength compared with reference B-form spectra. The helical repeats measured for the smaller fragments indicate that the helix winding angle can account for these features in the CD spectra. We suggest that G:C-rich boxes that punctuate not only the TFIIIA binding site but the whole 5 S gene are responsible for the conformational perturbation manifest in the CD spectra and may play a role in the recognition of the DNA by the factor. The spectrum of the gene is unchanged in the presence of TFIIIA, indicating that the structural heterogeneity of the DNA persists upon complex formation. The CD spectra of native TFIIIA.5 S rRNA particles isolated from oocytes and of particles reconstituted in vitro are identical and only moderately different from the spectrum of free 5 S rRNA, suggesting that the protein effects only limited changes in the secondary and/or tertiary structure of the RNA. The helical structure of the two binding sites is discussed with respect to a common mode of interaction of TFIIIA with DNA and RNA.