Poly (dA)· poly (dT) tracts are common and often found upstream of genes in eukaryotes. It has been suggested that poly (dA)· poly (dT) promotes transcription in vivo by affecting nucleosome formation. On the other hand, in vitro studies show that poly (dA)· poly (dT) can be easily incorporated into nucleosomes. Therefore, the roles of these tracts in nucleosome organization in vivo remain to be established. We have developed an assay system that can evaluate nucleosome formation in yeast cells, and demonstrated that relatively longer tracts such as A 15 TATA 16 and A 34 disrupt an array of positioned nucleosomes, whereas a shorter A 5 TATA 4 tract is incorporated in positioned nucleosomes of yeast minichromosomes. Thus, nucleosomes are destabilized by poly (dA)· poly (dT) in vivo in a length-dependent manner. Furthermore, in vivo UV footprinting revealed that the longer tracts adopt an unusual DNA structure in yeast cells that corresponds to the B′ conformation described in vitro. Our results support a mechanism in which a unique poly (dA)· poly (dT) conformation presets chromatin structure to which transcription factors are accessible.