The DNA in eukaryotic cells is organized into loop domains that are 25 to 100 kilobases long and attached at their bases to the nuclear matrix. This organization plays major roles in DNA replication and transcription. We examined changes in DNA loop structure of the 5S rDNA gene cluster in the Syrian golden hamster as a function of cellular differentiation by direct visualization with fluorescent in situ hybridization. The 5S rDNA cluster is large enough to encompass more than one loop domain but small enough that individual loop domains can still be resolved. We found that the sizes of the 5S rDNA loops are much smaller, and that the numbers of loops per locus are larger, in all pluripotent cell types than they are in adult somatic tissue. Within the pluripotent spermatogenic cell lineage, the loop domain organization was cell specific. The loop size decreased during the early stages of spermatogenesis but did not change during spermiogenesis, suggesting that DNA loop structure is independent of the chromatin condensation that occurs when protamines replace histones. In early embryonic cells, the loop structure remained small, but in differentiated somatic cells, it became much larger. We suggest that these changes in the 5S rDNA loop domain structure may be related to the maintenance or loss of developmental potential.