Flow (shear stress)-mediated outward remodeling of resistance arteries is a key adaptative process [1], which declines with age [2]. We recently demonstrated the essential role of 17beta-estradiol in flow-mediated remodeling of resistance arteries in vivo [3]. We now sought to assess the impact of age and timing of estrogen treatment after ovariectomy for better efficacy on this process. Rats of various ages (3 to 18 months) were submitted to surgery in order to increase locally blood flow in one mesenteric artery in vivo [4, 5], and the arteries, were collected 2 weeks later. Arterial diameter increased by 27% in high flow arteries of 3-months old male and female rats compared to normal flow vessels. In 12-month old rats, flow-mediated remodeling was absent in males, while it still reached 21% in females. Ovariectomy (3 weeks of estrogen deprivation) of 3, 9 and 12 month-old rats abolished flow-mediated remodeling, that was restored by immediate 17beta-estradiol replacement. Nevertheless, this permissive effect of E2 was attenuated or even abrogated if 17beta-estradiol replacement was delayed by 3 to 9 months respectively. This absence of remodeling was associated with reduction in both estrogen receptor alpha and endothelial nitric oxide (NO) synthase expression levels, associated with reduced NO-dependent dilation in high-flow mesenteric resistance arteries. Thus, long deprivation of 17beta-estradiol leads to a rapid decline in FMR, that can be prevented by early exogenous 17beta-estradiol supplementation, while delayed substitution of 9 months impairs 17beta-estradiol action. These data underline the importance of “timing” of 17beta-estradiol effect on this vascular process.