The genes encoding 18S, 5.8S, and 28S ribosomal RNA (rRNA) are tandemly repeated at the nucleolus organizer region (NOR). The NORs in the chicken map to one pair of microchromosomes. A line of chickens that contains individuals that are either disomic, trisomic, or tetrasomic for this chromosome, and have two, three, or four nucleoli and NORs, per cell, respectively, has been described previously. Aneuploid animals display a proportional increase in the rRNA gene copy number per cell. But, despite an increase in rDNA dosage, the levels of mature rRNA are regulated to normal levels in cells from aneuploid chickens (Muscarella, D.E., V.M. Vogt, and S.E. Bloom, 1985, J. Cell Biol., 101:1749-1756). This paper addresses the question of how regulation of mature rRNA synthesis occurs in cells with elevated levels of rDNA. An analysis of rRNA transcription in chicken embryo fibroblasts (CEFs) revealed that the relative rates of rRNA synthesis and processing and the amounts of precursor rRNA per cell are similar for all three genotypes. A comparison of chromatin structure, as determined by sensitivity of rDNA in nuclei from CEFs to digestion by DNase I, revealed that some of the rRNA genes from aneuploid cells are more resistant to digestion than corresponding sequences in the disomic cells. A determination of the distribution of topoisomerase I on rDNA has also been performed using the compound camptothecin, which introduces single- and double-strand breaks in topoisomerase-DNA complexes. Quantitation of camptothecin-induced cleavages revealed that a larger proportion of the rRNA genes in aneuploid cells was resistant to cleavage than in disomic cells, and therefore have no detectable amounts of topoisomerase I. These results suggest that the regulation of rRNA synthesis in CEFs with elevated levels of rDNA is achieved by the use of a subset of the rRNA genes.