<jats:p> Venous complications have been implicated in the adverse effects of hormone replacement therapy. This study investigated acute effects of the natural estrogen, 17β-estradiol, on function, estrogen receptors/GPR30 expression, and kinase activation in vascular rings and cultured smooth muscle cells from arteries and veins of patients with coronary artery disease. Changes in vascular tone of internal mammary arteries and saphenous veins exposed to the steroid were recorded. 17β-Estradiol caused concentration-dependent, endothelium-independent relaxation in arteries ( <jats:italic>P</jats:italic> <0.05 versus solvent control) but not in veins ( <jats:italic>P</jats:italic> not significant). 17β-Estradiol enhanced contractions to endothelin-1 in veins but not in arteries. The novel membrane estrogen receptor GPR30 was detected in both vessels. Moreover, gene expression of estrogen receptor β was 10-fold higher than that of estrogen receptor α or GPR30 ( <jats:italic>P</jats:italic> <0.05). Expression of all 3 of the receptors was reduced after exposure to 17β-estradiol in arteries but not in veins ( <jats:italic>P</jats:italic> <0.05). Basal phosphorylation levels of extracellular signal-regulated kinase were higher in venous than in arterial smooth muscle cells and were increased by 17β-estradiol in arterial cells only. In summary, this is the first study to report that, in human arteries but not in veins, 17β-estradiol acutely affects vascular tone, estrogen receptor expression, including GPR30, and extracellular signal-regulated kinase phosphorylation. These data indicate that effects of natural estrogens in humans differ between arterial and venous vascular beds, which may contribute to the vascular risks associated with menopause or hormone therapy. </jats:p>