Estrogen replacement therapy has been shown to attenuate atherogenesis, although the mechanisms for this effect are incompletely defined. Previously, we showed that 17-␤ estradiol (estradiol) attenuated oxidant stress-induced increases in vascular low density lipoprotein (LDL) accumulation. It was unclear whether estradiol's effect was imparted on the lipoprotein particle or the artery wall. To examine this, we chronically treated rats with the following sex hormones: low estradiol, high estradiol, progesterone, low estradiol ؉ progesterone, placebo, or control. Carotid arteries (n ‫؍‬ 8/group) were isolated and perfused with fluorescently labeled LDL. Rates of LDL accumulation were measured before and after treatment with 10 ng/ml tumor necrosis factor-␣ (TNF) using quantitative fluorescence microscopy. We observed a 50% decrease in basal LDL accumulation rates ( P Ͻ 0.01) and a 25% decrease in endothelial layer permeability ( P Ͻ 0.01) in arteries from estradiol-treated animals. There was no effect of hormone replacement on rate of TNF-induced LDL accumulation ( P ‫؍‬ 0.451), while incubation of LDL with 65 pg/ml estradiol attenuated the TNF effect ( P Ͻ 0.01). These experiments suggest two independent mechanisms of anti-atherogenic protection by estradiol: 1 ) decreased endothelial layer permeability; and 2 ) incorporation of estradiol into the LDL particle and prevention of LDL binding to the artery wall. -