Mild hyperhomocysteinemia is a risk factor for both ischaemic heart disease and venous thromboembolism. The effects of transdermal estrogen replacement therapy (ERT) on homocysteine metabolism in postmenopausal women have scarcely been investigated. This clinical trial aimed to estimate the effects of combined hormone replacement therapy on the fasting total homocysteine levels according to the estrogen route of administration. We enrolled 196 postmenopausal women, who were randomly allocated to receive on a continuous basis either 1mg of 17 beta-estradiol orally (n = 63) or 50 microg transdermally (n = 68) per day, both combined with a daily intake of 100 mg progesterone, or placebo (n = 65) over a period of 6 months. Neither oral nor transdermal ERT significantly affected total plasma homocysteine levels or red-blood cell folate levels. However, oral ERT significantly decreased plasma vitamin B12 levels compared to placebo (mean relative variation difference over 6 months between oral ERT and placebo: -11.7% (95%CI, -21 to -2%) whereas transdermal ERT did not display any significant effects. Our data show that transdermal ERT as well as low dose of oral ERT does not significantly affect the homocysteine metabolism. This finding does not support a role for transdermal estrogen in the prevention of ischaemic heart disease in postmenopausal women.