The mechanisms that control inactivation of ribosomal gene (rDNA) transcription during mitosis is still an open question. To investigate this fundamental question, the precise timing of mitotic arrest was established. In PtK1 cells, rDNA transcription was still active in prophase, stopped in prometaphase until early anaphase, and activated in late anaphase. Because rDNA transcription can still occur in prophase and late anaphase chromosomes, the kinetics of rDNA condensation during mitosis was questioned. The conformation of the rDNA was analyzed by electron microscopy from the G2/M transition to late anaphase in the secondary constriction, the chromosome regions where the rDNAs are clustered. Whether at transcribing or non-transcribing stages, non-condensed rDNA was observed in addition to axial condensed rDNA. Thus, the persistence of this non-condensed rDNA during inactive transcription argues in favor of the fact that mitotic inactivation is not the consequence of rDNA condensation. Analysis of the three-dimensional distribution of the rDNA transcription factor, UBF, revealed that it was similar at each stage of mitosis in the secondary constriction. In addition, the colocalization of UBF with non-condensed rDNA was demonstrated. This is the first visual evidence of the association of UBF with non-condensed rDNA. As we previously reported that the rDNA transcription machinery remained assembled during mitosis, the colocalization of rDNA fibers with UBF argues in favor of the association of the transcription machinery with certain rDNA copies even in the absence of transcription. If this hypothesis is correct, it can be assumed that condensation of rDNA as well as dissociation of the transcription machinery from rDNA cannot explain the arrest of rDNA transcription during mitosis. It is proposed that modifications of the transcription machinery occurring in prometaphase could explain the arrest of transcription, while reverse modifications in late anaphase could explain activation.