To investigate mechanisms of chromatin remodeling, we have examined the fate of a single nucleosome core within a spaced nucleosome array upon the binding of transcription factors. GAL4 binding to this nucleosome within an array resulted in the establishment of DNase I hypersensitivity adjacent to the bound factors mimicking in vivo hypersensitive sites. The positions of adjacent nucleosomes were unchanged upon GAL4 binding, suggesting that histone octamer sliding did not occur. In addition, novel assays were used to determine whether the histones remained present during factor binding. GAL4 binding alone did not independently dislodge or move the underlying histones, which remained in a ternary complex with the bound GAL4. GAL4 binding did, however, specifically predispose the histones contained in this nucleosome to displacement in trans. Addition of the histone binding protein, nucleoplasmin, mediated the displacement of the core histones in the GAL4-bound nucleosome, resulting in the formation of a nucleosome-free region. These data illustrate trans-displacement of histones as one mechanism for transcription factor-targeted generation of a nucleosome-free region in chromatin. They also illustrate the limitations of nuclease digestions in analyzing changes in chromatin structure and provide important mechanistic details beyond the basic phenomenon of DNase I hypersensitivity.