Abstract Body: Endothelial cell dysfunction is a central mechanism underlying the increased risk and prevalence of cerebrovascular accidents (CVAs) with aging. However, the direct and indirect impact of aging on endothelial cell biology remain complex and not fully understood. Circulating endothelial cell-derived microvesicles (EMVs) have emerged as both a biomarker and active contributor to vascular health and disease. Notably, EMVs have been implicated in the pathogenesis of cerebrovascular dysfunction, contributing to increased stroke risk and severity. The aim of this study was to determine, in vitro, the effect of circulating EMVs isolated from young and older adults on brain endothelial cell nitric oxide (NO) and endothelin (ET)-1 production. Reduced brain endothelial cell production of NO and increased ET-1 production have been linked to the pathogenesis of ischemic stroke. Twenty-two healthy, non-obese, normotensive, sedentary adults were studied: 10 young (6M/4F; age: 25±1 yr, BMI: 24.4+0.8 kg/m2) and 12 older (7M/5F; 64±2 yr; 23.4+0.7 kg/m2). EMV identification (CD144+) and isolation from peripheral blood were performed by flow cytometry. Human cerebral microvascular endothelial cells (hCMECs) were cultured and separately treated with EMVs from each subject. Circulating EMV concentrations were significantly higher in the older vs young adults (156±22 vs 91±8 EMV/μL). Phosphorylation is the primary posttranslational modification regulating endothelial nitric oxide synthase (eNOS) enzyme activity. Phosphorylation of Ser1177 confers the greatest activation of eNOS; whereas, phosphorylation at Thr495 reduces eNOS activation. Expression of p-eNOS (Ser1177) was lower (45.6±2.2 vs 70.5±4.8 AU; P<0.0001) and p-eNOS (Thr495) higher (39.9±2.6 vs 30.2±1.9 AU; P=0.009) in cells treated with EMVs from older vs young adults. Concordantly, NO production was markedly lower (5.5±0.3 vs 7.5±0.4 μmol/L; P<0.04) in cells treated with EMVs from older adults. There was no significant difference in the expression of Big ET-1 (72.9±4.4 vs 64.9±4.8 AU), endothelin converting enzyme (127.3±6.6 vs 131.1±5.3 AU) or ET-1 production (24.1±7.1 vs 19.6±1.5 pg/mL) in hCMECs treated with EMVs from older adults. In summary, EMVs from older adults reduced brain endothelial cell eNOS activation and NO production but not ET-1 production. Circulating EMVs may contribute to the increased risk of ischemic stroke with aging by impairing brain endothelial cell NO production.