The total genomic DNA methylation, i.e., the percentage of methylated cytosines, was measured in 20 cell lines derived from different types of human tumors. The measurements were obtained by cation-exchange liquid chromatography of bases released by formic acid hydrolysis. These experiments were done to determine if altered methylation is a prevalent and large defect in oncogenic transformation. A majority of the tumor cells measured had decreased levels of methylated DNA in comparison to our laboratory's and other laboratories' published measurements of normal cells and tissues. In fact, tumor cell DNA ranged as low as 1.2% of cytosines methylated compared to a value of 3% or more for normal cells and tissues. HpaII and MspI DNA restriction enzyme analysis confirmed for all tumor cell lines tested that their DNA was hypomethylated in comparison to the DNA from normal diploid fibroblasts tested. The results obtained by liquid chromatography and restriction enzyme analysis were strikingly similar. The reduced methylation of the tumor and DNA correlated with the recent observation of other laboratories that individual genes are undermethylated in human cancer cells and that a number of different carcinogens can lower DNA methylation directly.