Objective: This study tested, in ovariectomized rats, whether (1) adventitial activation plays a role in the vascular injury response, and (2) inhibition of adventitial activation and the subsequent wave of cell proliferation moving from adventitia to neointima contributes to the estrogen-induced attenuation of neointima formation in balloon injured carotid arteries. Methods: Ovariectomized Sprague-Dawley rats were treated with either 17β-estradiol or vehicle beginning 72 h prior to balloon injury of the right common carotid artery and were sacrificed at 0, 3, 7, 14 and 28 days after injury. BrdU was administered 18 h and 12 h prior to sacrifice in order to quantitate mitotic activity in adventitia, media and neointima of the damaged vessel at specified times post injury. Results: Adventitial activation, evidenced by positive BrdU staining, was evident on the day of injury, peaked on day 3 and was resolved by day 7, thus preceding neointima formation. Numbers of BrdU labeled cells in adventitia on day 3 were significantly reduced in estrogen treated rats compared to controls. BrdU labeled cells were undetectable in media on the day of injury, appeared at day 3 and disappeared by day 14. Neointima appeared at day 7 and increased in area throughout the period of observation. Neointimal area and numbers of BrdU labeled cells in neointima were significantly reduced in estrogen treated rats compared to controls. These findings suggest that there is a wave of cell proliferation moving in an adventitia-to-lumen direction following endoluminal injury of the rat carotid artery and that estrogen modulates this proliferative response to injury. Conclusion: These results support the hypothesis that adventitial activation contributes to the vascular injury response and that estrogen reduces this contribution. Copyright (C) 1999 Elsevier Science B.V.