This protocol describes a method of assaying nucleosome positions in S. cerevisiae by nuclease protection and quantitative PCR (Q-PCR). Briefly, yeast cells are spheroplasted, treated with micrococcal nuclease, and single-nucleosome length DNA is gel-isolated and quantitated by Q-PCR using overlapping primer pairs tiling the length of a region of interest. In the accompanying Nature paper, this technique is used to map the promoter nucleosome positions of several genes in the phosphate starvation response (PHO) pathway in order to determine the accessibility of Pho4 (transcription factor) binding sites. While this protocol has been used extensively with S. cerevisiae, it should, in principle, be readily adaptable for use in other yeast species.