Background - Mounting experimental evidence suggests that estrogen treatment protects against neointima formation in response to vascular injury in vivo. Previous studies have suggested that this process includes the activation and migration of adventitial fibroblasts. The present in vitro study was designed to establish a mechanism whereby estrogen attenuates migration of adventitial fibroblasts. Methods and Results - Primary cultures of vascular smooth muscle cells (VSMCs) and adventitial fibroblasts were derived from female Sprague-Dawley rats. Reverse transcriptase-polymerase chain reaction and Western blotting were used to determine that expression of the estrogen receptor (ER) was restricted to early-passage VSMCs. Migration of transduced (retrovirally mediated) fibroblasts was determined by counting the number of blue lacZ-expressing cells attached to Boyden-type chambers preconditioned under defined experimental conditions. Compared with growth medium alone, chambers treated with medium conditioned by VSMCs demonstrated a 2-fold increase in fibroblast migration, suggesting that VSMCs release soluble factor(s) competent to bind the Transwell membrane and promote fibroblast migration. In contrast, treatment of VSMCs with 17β-estradiol (10-9 to 10-7 mol/L) before preconditioning of the chamber induced a dose-dependent inhibition of fibroblast migration. Cotreatment of VSMCs with 17β-estradiol and the ER antagonist ICI-182780 (10-7 mol/L) blocked the inhibitory effect of estrogen on fibroblast migration. Conclusions - These observations suggest a novel mechanism of hormonal vasoprotection by which estrogen directly modulates VSMC expression of factor(s) controlling migration of adventitial fibroblasts via an ER-dependent mechanism.