5-hydroxymethylcytosine (5-hmC) has been recently discovered in mammalian DNA by two US groups (Kriaucionis & Heintz, Science, 2009 and Tahiliani et al. ,Science, 2009), however, its precise function has not yet been elucidated. This cytosine modification results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. 5-hydroxymethylcytosine may represent a new pathway to demethylate DNA involving a repair mechanism converting hmC to C and, as such open up entirely new perspectives in epigenetic studies. Since its discovery in neuronal Purkinje, granule and ES cells, studies of this new modified DNA base have been limited by the lack of high quality, validated tools and technologies that discriminate hydroxymethylation from methylation in regulating genome expression. Obtaining a specific assay for 5-hmC is particularly important since standard methods (eg. bisulfite sequencing) cannot distinguish between these two types of methylation.