DNA methylation has been viewed as a stable component of the epigenome, established during development and fixed thereafter. Here we have found that the DNA methylation pattern varies during a single cell cycle, with the global levels of DNA methylation decreased in G(1) and increase during S phase. There was little change in the DNA methylation levels in repetitive sequences throughout the cell cycle. However using a human CpG island microarray it was revealed that 174 CG-containing sequences were differentially methylated between G(1) and S. Seventy-five percent of all the variations in DNA methylation detected in unique sequences represented hypomethylation at G(0), with changes occurring in both CpG islands and non-CpG islands. This is the first demonstration of a dynamic DNA methylation pattern within a single cell cycle of a mature somatic cell. These data are important for our understanding of the stability of DNA methylation patterns in somatic cells.