In cancer, the extensive methylation found in the bulk of chromatin is reduced, while the normally unmethylated CpG islands become hypermethylated. Regions of solid tumors are transiently and/or chronically exposed to ischemia (hypoxia) and reperfusion, conditions known to contribute to cancer progression. We hypothesized that hypoxic microenvironment may influence local epigenetic alterations, leading to inappropriate silencing and re-awakening of genes involved in cancer. We cultured human colorectal and melanoma cancer cell lines under severe hypoxic conditions, and examined their levels of global methylation using HPLC to quantify 5-methylcytosine (5-mC), and found that hypoxia induced losses of global methylation. This was more extensive in normal human fibroblasts than cancer cell lines. Cell lines from metastatic colorectal carcinoma or malignant melanoma were found to be markedly more hypomethylated than cell lines from their respective primary lesions, but they did not show further reduction of 5-mC levels under hypoxic conditions. To explore these epigenetic changes in vivo, we established xenografts of the same cancer cells in immune deficient mice. We used Hypoxyprobe to assess the magnitude of tissue hypoxia, and immunostaining for 5-mC to evaluate DNA methylation status in cells from different regions of tumors. We found an inverse relationship between the presence of extensive tumor hypoxia and the incidence of methylation, and a reduction of 5-mC in xenografts compared to the levels seen in the same cancer cell lines in vitro, verifying that methylation patterns are also modulated by hypoxia in vivo. This suggests that epigenetic events in solid tumors may be modulated by microenvironmental conditions such as hypoxia.