Peng Jin, Bing Ren, Chuan He and colleagues report a method called Tet-assisted bisulfite sequencing (TAB-seq) to map 5-hydroxylmethylcytosine (5hmC) across the genome at single-base resolution (Cell 149, 1368–1380, 2012). The method can discriminate 5hmC from 5-methylcytosine (5mC), as 5hmC is selectively protected by β-glucosyltransferase, thereby rendering 5mC uniquely susceptible to TET-mediated oxidation to 5-carboxylcytosine. Bisulfite treatment then converts cytosine and 5-carboxylcytosine to uracil and 5-carboxyluracil, respectively, which are read as thymine during sequencing, whereas β-glucosyl-5-hydroxylmethylcytosine is read as cytosine. The authors applied TAB-seq to mouse and human embryonic stem cell genomes and found that 5hmCs are present in clusters and that 5mC and 5hmC can coexist at the same cytosine in a population of cells. Unlike 5mC, 5hmC is asymmetric at CG sequences and shows a bias for G-rich sequences. The authors noted enrichment of 5hmC immediately adjacent to transcription factor–binding sites and also found that approximately half of 5hmCs are located in distal regulatory elements, such as p300-binding sites, enhancers, CTCF-binding sites and DNase I hypersensitive sites.