The structures of non-coding and coding strands in box C of the internal control region (ICR) of Xenopus laevis somatic 5S RNA gene have been examined by circular dichroism (CD) and Raman spectroscopy in the absence and presence of the third zinc finger of transcription factor IIIA (TFIIIA), which binds to the ICR. The non-coding strand exhibits CD signals assignable to a hairpin and an unfolded structure. The presence of the hairpin structure is supported by Raman spectra, gel electrophoresis, and nucleotide deletion experiments. Binding of the zinc finger to the non-coding strand increases the CD signal of hairpin structure, indicating stabilization of the hairpin structure by the zinc finger. In contrast, the corresponding coding strand remains unfolded even in the presence of the zinc finger. The TFIIIA-ICR complex is not only required for initiation of transcription but also lasts during many rounds of transcription of the 5S RNA gene including the ICR (Bogenhagen et al., Cell 28 (1982) 413). TFIIIA may play a role in promoting the transcription by maintaining the unwound non-coding strand in the hairpin structure and leaving the coding strand available for transcription by RNA polymerase.