The positions of nucleosomes along genomic DNA play a role in defining patterns of gene expression and chromatin organization. Determination of nucleosome positions in vivo and in vitro, as revealed by the locations of histones on DNA, has provided insight into mechanisms of nucleosome sliding, spacing, assembly, and disassembly. Here, we describe methods for the in vitro determination of histone-DNA contacts at base-pair (bp) resolution. The protocol involves the labeling of histones with ortho-phenanthroline (OP), site-specific cleavage of nucleosomal DNA, and processing and analysis of the resulting DNA fragments. This methodology provides an efficient and high-resolution means for studying kinetics and behavior of enzymes that alter nucleosome structure and/or positioning, and can be used to identify preferred distributions of nucleosomes on natural DNA sequences. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Cysteine-specific chemical modification of folded histones with ortho-phenanthroline (OP) Basic Protocol 2: Nucleosome sliding assay adapted for OP mapping of histone-DNA contacts Basic Protocol 3: OP-mediated cleavage, processing, and analysis of DNA fragments using a sequencing gel Support Protocol 1: Preparation of dideoxy sequencing ladders Support Protocol 2: Preparation and running of a denaturing DNA sequencing gel.