Nucleosomes were reconstituted from 170 bp long fragments of 5S rDNA and an optimal positioning sequence, the Selex 601, with recombinant histories. In free-solution single pair Forster resonance energy transfer (spFRET) measurements of the distance between fluorescently labeled bases in the nucleosomal DNA, the samples exhibited structural diversity. The structural heterogeneity correlated with the stability of the complexes and depended on the DNA sequence and histone acetylation. The stability of the nucleosomes was assessed via dilution-driven disruption: histone acetylation decreased nucleosome stability. The spFRET experiments used a new approach for data acquisition and analysis that we term "deliberately detuned detection" (D-3). This permits the separation of subpopulations in the samples even for the low-FRET regime characteristic for the linker-DNA labeled nucleosomes. Thus, it became possible to study in more detail histone acetylation- and salt-dependent structural variations using either end-or internally labeled DNAs on the nucleosome. We found that the distance distribution of the fluorophore pairs on the linker DNA ends was much more sensitive to historic acetylation or sequence variation than that of labels on the internal part of the DNA, which was more tightly associated with the histone core. spFRET on freely diffusing nucleosomes allows us therefore to localize the influence of histone modifications and DNA sequence variations on the nucleosome structure and dynamics.