Abstract—The vascular consequences of estrogen treatment may be driven by its initiation timing. We tested the hypothesis that the duration of ovarian hormone deprivation before estrogen reintroduction affects the role of estrogen as mediator of endothelial function and vascular relaxation in nondiseased vessels. Rats were ovariectomized and implanted with 17-estradiol (E₂) or oil capsules 1, 4, and 8 months after surgery. After the longest hypoestrogenicity period, acetylcholine-mediated aortic relaxation was attenuated and insensitive to E₂ administration despite endothelial integrity. Whereas no rapid vasorelaxant responses were elicited by an estrogen receptor (ER) –selective agonist, responses to E₂ and an ER selective agonist waned postovariectomy at any given time and were restored by E₂ treatment after 1 and 4 months but not 8 months postovariectomy. Accordingly, endothelial ER mRNA and protein expression declined 6-fold after prolonged hypoestrogenicity and was restored by estrogen replacement starting 1 month but not 8 months postovariectomy. Furthermore, the amount of active phosphorylated endothelial NO synthase rose significantly after E₂ replacement after 1 and 4 months but not 8 months postovariectomy. The present findings document that the functional impairment of the ER/endothelial NO synthase signaling network after an extended period of hypoestrogenicity was not restored by E₂ administration, providing experimental support to early initiation of estrogen replacement with preferential ER targeting to improve cardiovascular outcomes.