Medroxyprogesterone acetate (MPA) is widely known for its use in combination hormone therapy for postmenopausal women. However, MPA is also commonly used in young women for contraception and treatment of a number of gynecological conditions. Despite its widespread use, the cardiovascular effects of MPA in young women are unclear. Therefore, the purpose of this study was to determine the acute effects of MPA when used in combination with estradiol on biomarkers of cardiovascular risk in young women. We suppressed endogenous estrogens and progesterone in 10 premenopausal women using a gonadotropin-releasing hormone antagonist (GnRHa) for 10 days. On day 4 of GnRHa, subjects received 0.1 mg estradiol (GnRHa+E2), and on day 7 added 5 mg MPA (GnRHa+E2+MPA). Endothelium-dependent vasodilation and endothelium-independent vasodilation of the brachial artery, lipids, homocysteine, high sensitivity C-reactive protein, and endothelin-1 were assessed during treatment with GnRHa, GnRHa+E2, and GnRHa+E2+MPA. Four additional subjects were tested to validate the efficacy of the GnRHa model and confirm the findings. Endothelium-dependent vasodilation was greater during GnRHa+E2 than GnRHa or GnRh+E2+MPA (p=0.006). Endothelin-1 was lower during GnRHa+E2 than GnRHa alone (p=0.039). Endothelin-1 increased with the addition of MPA and was not significantly different from GnRHa alone. There were no differences in the other markers of cardiovascular risk between hormone treatment days. These data suggest that acute MPA administration negates the beneficial effects of estradiol on endothelium-dependent vasodilation in young women. In addition, these data suggest estradiol decreases endothelin-1 concentrations and the addition of MPA may counteract the effect of estradiol on endothelin-1.