The internal promoter of the Xenopus laevis oocyte-type 5S RNA gene is preferentially cleaved by S1 and Bal-31 nucleases in plasmid DNA. S1 nuclease sensitivity is largely dependent on supercoiling; however, Bal-31 cleaves within the 5S RNA gene in linear as well as in supercoiled DNA. The S1 nuclease-hypersensitive site is centered at position +48-52 of the gene at the 5' boundary of the promoter. A DNAase I-hypersensitive site is induced at this position upon binding of the transcription factor, TFIIIA, specific for the 5S RNA gene. The somatic-type 5S RNA gene promoter is not preferentially cleaved by S1 nuclease or Bal-31 nuclease in supercoiled DNA, nor does TFIIIA induce a DNase I site at position +50. This differential promoter response may be related to a 4-fold difference in TFIIIA affinity between the oocyte and somatic 5S RNA genes.