Full-length 66-kDa estrogen receptor⍺ (ER⍺) stimulates target gene transcription through two activation functions (AFs), AF-1 in the N-terminal domain and AF-2 in the ligand binding domain. Another physiologically expressed 46-kDa ER⍺ isoform lacks the N-terminal A/B domains and is consequently devoid of AF-1. Previous studies in cultured endothelial cells showed that the N-terminal A/B domain might not be required for estradiol (E2)-elicited NO production. To evaluate the involvement of ER⍺AF-1 in the vasculoprotective actions of E2, we generated a targeted deletion of the ER⍺ A/B domain in the mouse. In these ER⍺AF-1zero mice, both basal endothelial NO production and reendothelialization process were increased by E2 administration to a similar extent than in control mice. Furthermore, exogenous E2 similarly decreased fatty streak deposits at the aortic root from both ovariectomized 18-weekold ER⍺AF-1+/+LDLR-/- (low-density lipoprotein receptor) and ER⍺AF-1zeroLDLR-/- mice fed with a hypercholesterolemic diet. In addition, quantification of lesion size on en face preparations of the ic tree of 8-month-old ovariectomized or intact female mice revealed that ER⍺AF-1 is dispensable for the atheroprotective action of endogenous estrogens. We conclude that ER⍺AF-1 is not required for three major vasculoprotective actions of E2, whereas it is necessary for the effects of E2 on its reproductive targets. Thus, selective ER modulators stimulating ER⍺ with minimal activation of ER⍺AF-1 could retain beneficial vascular actions, while minimizing the sexual effects.