5-hydroxymethylcytosine (5hmC) was recently identified as an abundant epigenetic mark in mammals. Subsequent research has implicated 5hmC in normal mammalian development and disease pathogenesis in humans. Many of the techniques commonly used to assay for canonical 5-methylcytosine (5mC) cannot distinguish between 5hmC and 5mC. The development of antibodies specific to 5hmC has allowed for specific enrichment of DNA fragments containing 5hmC. Hydroxymethylated DNA immunoprecipitation (hmeDIP) has become an invaluable tool for determining both locus-specific and genome-wide profiles of 5hmC in mammalian DNA. Here, we describe the use of hmeDIP to characterize the relative abundance of 5hmC at loci in mammalian DNA.