Polypurine tracts are important elements of eukaryotic promoters. They are believed to somehow destabilize chromatin, but the mechanism of their action is not known. We show that incorporating an A₁₆ element at an end of the nucleosomal DNA and further inward destabilizes histone-DNA interactions by 0.1 ± 0.03 and 0.35 ± 0.04 kcal mol⁻¹, respectively, and is accompanied by 1.5- ± 0.1-fold and 1.7- ± 0.1-fold increases in position-averaged equilibrium accessibility of nucleosomal DNA target sites. These effects are comparable in magnitude to effects of A₁₆ elements that correlate with transcription in vivo, suggesting that our system may capture most of their physiological role. These results point to two distinct but interrelated models for the mechanism of action of polypurine tract promoter elements in vivo. Given a nucleosome positioned over a promoter region, the presence of a polypurine tract in that nucleosome's DNA decreases the stability of the DNA wrapping, increasing the equilibrium accessibility of other DNA target sites buried inside that nucleosome. Alternatively (if nucleosomes are freely mobile), the presence of a polypurine tract provides a free energy bias for the nucleosome to move to alternative locations, thereby changing the equilibrium accessibilities of other nearby DNA target sites.