The cytomegalovirus promoter is a very potent promoter commonly used for driving the expression of transgenes, though it gradually becomes silenced in stably transfected cells. We examined the methylation status of the cytomegalovirus promoter in two different cell lines and characterized its mechanisms of activation by dimethylsulfoxide and 5-Aza-2'-deoxycytidine. The cytomegalovirus promoter stably transfected into Chinese hamster ovary cells is suppressed by DNA methylation-independent mechanisms, which is different from the rat embryonic cardiomyoblast H9c2-Fluc.3 cells in which the cytomegalovirus promoter is silenced by methylation. Dimethylsulfoxide and 5-Aza-2'-deoxycytidine can activate the cytomegalovirus promoter in both cell types by overlapping mechanisms. Dimethylsulfoxide activates the cytomegalovirus promoter in Chinese hamster ovary cells by promoting histone acetylation and the activation of p38 mitogen-activated protein kinase and nuclear factor kappaB (NFkappaB) signaling pathways, while 5-Aza-2'-deoxycytidine increases histone acetylation and activates the nuclear factor kappaB but not the p38 mitogen-activated protein kinase pathway. In H9c2-Fluc.3 cells, both agents promote demethylation of the cytomegalovirus promoter, and enhance its activity exclusively through activation of the nuclear factor kappaB pathway and to a lesser extent of the p38 mitogen-activated protein kinase pathway. Our findings suggest that suppression and activation of the cytomegalovirus promoter are cell type-specific. These results may be used for developing strategies to enhance the expression of transgenes and the production of recombinant proteins encoded by transgenes controlled by a cytomegalovirus promoter.