Recent advances in experimental and computational methodologies are enabling ultra-high resolution genome-wide profiles of protein–DNA binding events. For example, the ChIP-exo protocol precisely characterizes protein–DNA cross-linking patterns by combining chromatin immunoprecipitation (ChIP) with 5′ → 3′ exonuclease digestion. Similarly, deeply sequenced chromatin accessibility assays (e.g. DNase-seq and ATAC-seq) enable the detection of protected footprints at protein–DNA binding sites. With these techniques and others, we have the potential to characterize the individual nucleotides that interact with transcription factors, nucleosomes, RNA polymerases and other regulatory proteins in a particular cellular context. In this review, we explain the experimental assays and computational analysis methods that enable high-resolution profiling of protein–DNA binding events. We discuss the challenges and opportunities associated with such approaches.