A new high-resolution approach for the genome-wide detection of DNase I hypersensitive sites (DHSs) can be applied to single cells. Approximately 317,000 unique reads and 38,000 DHSs, on average, were detected per single cell by single-cell DNase sequencing (scDNase-seq). The authors show that pooled DHSs of five single NIH3T3 cells correlate significantly with those of 1,000 cells, and single-cell DHS patterns are highly reproducible between individual cells. Single-cell DHSs predicted enhancers that regulate cell-specific gene expression programmes, and cell-to-cell variations of individual DHSs were predictive of gene expression. scDNase-seq of cells dissected from formalin-fixed paraffin-embedded tissue slides obtained from patients with thyroid cancer identified thousands of tumour-specific DHSs.