Whilst evidence suggests that epigenetic modifications of DNA and histone interact to modulate gene expression, the precise sequence and extent of this interaction is unclear and contrasting reports exist. We have previously observed changes in DNA methylation and microRNA expression that reflect the molecular biology of UCC and are associated with the clinical phenotype of tumors. In particular, DNA methylation appears a common carcinogenic event that occurs early in the disease pathway and an independent predictor of tumor progression. Whilst indicating an important role for epigenetic gene regulation in UCC, these studies were limited to only one mechanistic tier of control and did not analyze histone alterations. To gain a more in depth knowledge of repressive epigenetic gene regulation in UCC, we have now profiled H3K9m3 and H3K27m3 in normal and malignant urothelial cells. We matched these profiles to those for 5methylcytosine and gene expression. We hypothesized that differences represent pro-carcinogenic events within the urothelium. Here we have produced integrated epigenomic maps for two UCC cell lines and non-transformed normal urothelial cells. The latter are cultured expansions taken from disease free patients that grow as sheets of Dimesna histologically normal appearing urothelial cells for 7–8 passages before senescence. Our experimental data reveal insights into the epigenetic control of gene expression in the biology of UCC and have specifically identified genes potentially involved in urothelial carcinogenesis. Whilst this association may be because both marks are negatively correlated to gene expression, it could reflect a direct causation. Whilst our methodology has identified associations between epigenetic marks and gene expression, we did not examine this cause or direction. Data to examine this relationship may be obtained from the literature. Schlesinger et al. identified that genes silenced in cancer are initially associated with H3K27m3. This mark is maintained by EZH2, which recruits DNA methyl transferases and these in turn methylate previously unmethylated cytosine residues. Rush et al. then identified that EZH2 specifically recruits DNMT3a, but that this alone is insufficient for de novo methylation,Diatrizoic acid suggesting a need for additional events. Support for this order of events could be found in silenced genes with H3K27m3 but not DNA methylation. We identified many such examples, as did Kondo et al.. The clearest observations from our data are those regarding the integrated nature of epigenetic gene regulation in the urothelium. Gene enrichment pathway analysis suggested diverse roles for the three epigenetic marks. For example, genes marked by H3K9m3 were involved in cellular metabolism and the response to external stimuli. These important cellular pathways need to remain constant within a cell and not vary with transformation. In contrast, genes marked with H3K27m3 appeared carcinogenic in function and were involved with cell division, chromatin assembly, regulation of transcription, the induction of apoptosis and cell migration. Genes with only 5 mC enrichment were clustered into pathways dealing with the response to stress and oxidation, cell morphogenesis and movement. These pathways represent a mixture of those important for cell homeostasis and those involved in cancer. Given our findings, one would suspect that the pro-carcinogenic pathways associated with 5 mC are those contributing to malignant transformation. To assess the validity of the genes identified using cell lines, we examined their expression in 150 normal and malignant urothelial samples. Our epigenetic panel correctly estimated the expression of 65% of genes and stratified the tissues according to the presence and phenotype of cancer.