Mammalian DNA methylathion is a chemical reaction catalyzed by DNA methyltransferases (DNMTs) and involves the addition of a methyl group from the methyl donor SAM to the carbon 5 position of cytosine (C) in a CpG dinucleotide. Specifically, DNA methylation is essential for normal development and is involved in numerous key mechanisms such as genomic imprinting, X-chromosome inactivation, suppression of repetitive elements and may be involved in the regulation of single-copy gene expression. In the human genome the majority of CpGs are methylated whereas regions with high density of CpG sites, termed CpG islands and often co-localized within gene promoters, are typically free of this mark. Recently, a new modified cytosine, 5-hydroxymhetylcytosine (5-hmC), was identified and found at significant levels in mouse brain and both mouse and human embryonic stem (ES) cells. The conversion of 5-mC to 5-hmC is catalyzed by the ten-eleven translocation (TET) proteins of the 2-oxoglutarate (2OG)-and Fe(II)-dependent oxygenase superfamily. Many studies were conducted since the identification of 5-hmC and significant levels of 5-mC hydroxylation were found in many other mouse and human tissues. Importantly, many of the techniques used for 5-mC detection, such as bisulphite sequencing and methyl-sensitive restriction digestion, are incapable of distinguishing between 5mC and 5hmC implying the necessity not only to develop techniques specific for 5-hmC characterization but also reevaluation of previously published 5mC data. The biological function of 5-hmC is unknown however many recent studies have suggested a role for 5-hmC as an intermediate of either passive or active demethylation. The majority of studies of 5- hmC and TETs have used mouse ES cells as model system. Therefore, very little is known about 5-hmC patterns and TET expression within and between normal tissues. During my PhD, I used the recently developed 5-hmC-specific antibody for tiling microarrays and 5hmC-qPCR to examine both global 5hmC content and locus-specific patterns of 5hmC in several normal human tissues and breast cancer. I found that global 5-hmC content is highly variable between tissues compared to global 5-mC content. Moreover, TETs genes are highly expressed in most of tissues tested. Importantly, both global 5-hmC content and TETs genes are rapidly and significantly reduced as consequence of adaptation of cells from normal human tissue to cell culture. Using the 5hmC-specific antibody for tiling microarrays and 5-hmC-qPCR to profile locus-specific patterns of 5hmC, I found that 5-hmC patterns are tissue-specific in human samples. In addition, comparing array data to RNA-seq data, 5- hmC was found to co-localize at gene bodies of active genes. Moreover, despite the global 5-hmC reduction in cell lines, 5-hmC content remains enriched in some specific loci. In summary, my results show that tissue type is a major modifier of both global and locus-specific 5hmC at genes in normal human tissues. Furthermore, I also show that both TET gene expression and 5hmC content are significantly reduced and 5-hmC profiles reprogrammed during the passage from tissues to cell culture.