Abstract: A canonical nucleosome architecture around promoters establishes the context in which proteins regulate gene expression. Whether gene regulatory proteins that interact with nucleosomes are selective for individual nucleosome positions across the genome is not known. Here, we examine on a genomic scale several protein-nucleosome interactions, including those that (1) bind histones (Bdf1/SWR1 and Srm1), (2) bind specific DNA sequences (Rap1 and Reb1), and (3) potentially collide with nucleosomes during transcription (RNA polymerase II). We find that the Bdf1/SWR1 complex forms a dinucleosome complex that is selective for the +1 and +2 nucleosomes of active genes. Rap1 selectively binds to its cognate site on the rotationally exposed first and second helical turn of nucleosomal DNA. We find that a transcribing RNA polymerase creates a delocalized state of resident nucleosomes. These findings suggest that nucleosomes around promoter regions have position-specific functions and that some gene regulators have position-specific nucleosomal interactions.