To evaluate the effect of estrogen therapy (ET) on endothelial nitric oxide (NO) production and in flow-mediated vasodilation (FMV). Randomized, crossover, double-blind, placebo-controlled study. Healthy postmenopausal women in an academic research environment. Forty postmenopausal women between 45 and 72 years of age. Women received ET or placebo during two periods of 12 weeks that were separated by 2 weeks of washout. Flow-mediated vasodilation, nitrite and nitrate, lipid profile, creatinine, and glucose were measured at weeks 12 and 24. Student's t or Wilcoxon tests were used for comparative analyses, and κ test and limit analysis determined variability. After placebo treatment, nitrate and nitrite mean concentration was 8.28 ± 1.17 mmol/L; it increased to 62.6 ± 12.82 mmol/L after ET. Percentage FMV was 18.8 ± 2.58 after the placebo period and did not change after ET (20.1 ± 1.92) in the whole sample, but in the subgroup (n = 15) of younger women (4550 years of age), percentage FMV increased from 13.6 ± 3.6 after the placebo period to 22.2 ± 3.5 after ET. An increase in plasma concentrations of nitrite and nitrate after ET was observed in all the women studied, but the improvement in FMV was observed only in the younger ones. These age-related differences in FMV in response to ET must be further investigated.