DNA methylation plays a central role in the control of gene expression during development and cell differentiation, thus DNA methylation and demethylation processes are expected to be strictly regulated during these events. We have explored the expression levels of the genes encoding DNA methylases, methyl-CpG binding proteins and demethylases during in vitro differentiation of human carcinoma colon cells (CaCO-2) used as a model system. The results show that the global DNA methylation pattern remains constant during CaCO-2 cells differentiation indicating that required genome methylation pattern in cell differentiation was already established in the seeded cells. On the contrary, the timing of expression of several of the explored genes is tightly regulated, suggesting they are involved in the regulation of the differentiation program. In particular, the timing of expression of DNMT3b and of MBD2b and 5-MCDG shows two peaks not observed in the time courses of the expression of other genes belonging to the same families. These events, not dependent on the cell cycle synchronization, have apparently no significant impact on the overall methylation status of the genome.