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Roots from Allium cepa L. (cv. Francesa) bulbs in which a maximum of two nucleoli per nucleus developed were selected for this study. Five rDNA clusters were detected by fluorescent in situ hybridization on chromosomal squashes (2n = 16) with a rhodamine-labelled wheat rDNA repeat. The rDNA clusters were located on four chromosomes: the largest cluster occurred on the small arm of a single homologue of the smallest pair 8. Its homologue showed two different small rDNA clusters, one near each telomere. The two homologues of the satellited chromosomes 6 also showed different rDNA contents, which were intermediate to those found in pair 8. The same five well-differentiated hybridization signals were observed in interphase cells that were inactive in transcription because they were in dormant roots, or in proliferating ones in which the synthesis of the large rRNA precursor was prevented. After multipolarizing agent was applied in anaphase followed by inhibition of cytokinesis, multinucleate autotetraploid cells were formed, which often contained more than four nucleoli. Thus, at least two of the three nucleolar organizer regions that consistently failed to develop a nucleolus in normal mononucleate cells were capable of developing nucleoli when segregated into different nuclei in multinucleate cells.