Background - Previous in vitro studies have suggested that estrogen attenuates the vascular injury response by modulating vascular smooth muscle cell (VSMC) expression of soluble factor(s) directing migration of adventitial fibroblasts. Previous in vivo studies have established a role for osteopontin (OPN) and its integrin receptors after vascular injury. In this study, we examined OPN expression in activated VSMCs, its modulation by estrogen, and its effects on adventitial fibroblast migration. In addition, the relative functional roles of β1- and β3-integrin-matrix interactions were examined. Methods and Results - Primary cultures of VSMCs and adventitial fibroblasts were derived from female Sprague-Dawley rats. Serum- activated VSMCs expressed high levels of OPN mRNA and secreted protein that was effectively inhibited by estrogen treatment (10-7 mol/L). Compared with VSMCs, fibroblasts expressed similar levels of integrins αν and β1 and higher levels of integrin-β3. Exogenous OPN (5.0 to 40 μg/mL) directed fibroblast migration in a dose-dependent fashion. Anti-β3-integrin antibody (F11) pretreatment markedly inhibited adventitial fibroblast migration directed by exogenous OPN or VSMC-conditioned medium in a dose-dependent manner. In contrast, anti-β1-integrin antibody (Ha2/5) did not affect fibroblast migration. Similarly, pretreatment with either linear or cyclic RGD peptides (10 to 1000 μmol/L) inhibited fibroblast migration directed by OPN or VSMC-conditioned medium in a dose-dependent manner. Conclusions - These observations suggest that estrogen indirectly attenuates integrin-β3- dependent adventitial fibroblast migration after inhibition of OPN expression in VSMCs.