In the present study, we hypothesized that estradiol, via its ability to vasodilate in an endothelium-dependent manner, might enhance vascular effects of insulin. Basal and insulin-stimulated peripheral blood flow and resistance, arterial stiffness, and glucose metabolism were determined in 27 healthy postmenopausal women before and after 12 weeks of treatment with either transdermal or oral estradiol or corresponding placebo preparations. Whole body insulin sensitivity was determined using the euglycemic insulin clamp technique (rate of continuous insulin infusion 1 mU/kg.min), forearm blood flow with a strain-gauge plethysmography, and arterial stiffness using pulse wave analysis. Estradiol therapy increased basal peripheral blood flow (1.5 +/- 0.1 vs. 1.9 +/- 0.1 mL/dL.min, 0 vs. 12 weeks; P: < 0.01), decreased peripheral vascular resistance (65 +/- 3 vs. 52 +/- 3 mm Hg/mL/dL.min, respectively; P: < 0.01), and diastolic blood pressure (78 +/- 2 vs. 75 +/- 2 mm Hg, respectively; P: < 0.05) but had no effect on large artery stiffness. Infusion of insulin did not acutely alter peripheral blood flow but diminished large artery stiffness significantly both before and after the 12-week period of estradiol therapy. No measure of acute insulin action (glucose metabolism, blood flow, or large artery stiffness) was altered by estradiol or placebo treatment. These data demonstrate that insulin and estradiol have distinct hemodynamic effects. Physiological doses of estradiol increase peripheral blood flow but have no effects on large artery stiffness, whereas physiological concentrations of insulin acutely decrease stiffness without changing peripheral blood flow. Putative vasculoprotection by estradiol is, thus, not mediated via alterations in arterial stiffness or insulin sensitivity.