The HMG box containing protein UBF binds to the promoter of vertebrate ribosomal repeats and is required for their transcription by RNA polymerase I in vitro. UBF can also bind in vitro to a variety of sequences found across the intergenic spacer in Xenopus and mammalian ribosomal DNA (rDNA) repeats. The high abundance of UBF, its colocalization with rDNA in vivo, and its DNA binding characteristics, suggest that it plays a more generalized structural role over the rDNA repeat. Until now this view has not been supported by any in vivo data. Here, we utilize chromatin immunoprecipitation from a highly enriched nucleolar chromatin fraction to show for the first time that UBF binding in vivo is not restricted to known regulatory sequences but extends across the entire intergenic spacer and transcribed region of Xenopus, human, and mouse rDNA repeats. These results are consistent with a structural role for UBF at active nucleolar organizer regions in addition to its recognized role in stable transcription complex formation at the promoter.