Objective: Methylation of the promoter region of the estrogen receptor gene alpha (ER α) occurs as a function of age in human colon, and results in inactivation of gene transcription. In this study, we sought to determine whether such age-related methylation occurs in the cardiovascular system, and whether it is associated with atherosclerotic disease. Methods: We used Southern blot analysis to determine the methylation state of the ER α gene in human right atrium, aorta, internal mammary artery, saphenous vein, coronary atherectomy samples, as well as cultured aortic endothelial cells and smooth muscle cells. Results: An age related increase in ER α gene methylation occurs in the right atrium (range 6 to 19%, R=0.36, P<0.05). Significant levels of ER α methylation were detected in both veins and arteries. In addition, ER α gene methylation appears to be increased in coronary atherosclerotic plaques when compared to normal proximal aorta (10±2% versus 4±1%, P<0.01). In endothelial cells explanted from human aorta and grown in vitro, ER α gene methylation remains low. In contrast, cultured aortic smooth muscle cells contain a high level of ER α gene methylation (19–99%). Conclusions: Methylation associated inactivation of the ER α gene in vascular tissue may play a role in atherogenesis and aging of the vascular system. This potentially reversible defect may provide a new target for intervention in heart disease.