Although the cardiovascular benefits of the hormone estrogen are at least, in part, mediated by its antiproliferative effect on vascular smooth muscle, its action on the migration of these cells is unknown. To explore this relationship, female rat aortic smooth muscle cells were grown in hormone-free medium, and the effect of various concentrations of beta-estradiol on directed cellular migration was measured in vitro using a microwell Boyden chamber apparatus. Migration of smooth muscle cells to the known chemoattractants platelet-derived growth factor, insulin-like growth factor-1, and fibronectin (all at peak doses for migratory activity) was attenuated by beta-estradiol (0.5 to 10 ng/ml) in a concentration-dependent manner relative to control cells treated with vehicle (0.01% ethanol). This response was insensitive to pretreatment with indomethacin and was stereospecific (17 alpha-estradiol lacked response). Like beta-estradiol, the synthetic estrogen diethylstilbestrol attenuated directed smooth muscle cell migration whereas the male hormone testosterone was ineffective. Additional studies showed that beta-estradiol-mediated suppression of migration was inhibited by the anti-estrogen ICI 164,384 and the gene transcription inhibitor actinomycin D. These are the first results demonstrating a reduction in directed smooth muscle cell migration by beta-estradiol. The mechanism of this estrogen-mediated response appears to involve conventional estrogen receptors.