The cardioprotective effects of estrogen in premenopausal women appear to be mediated in part by effects on endothelial cell biology. A major determinant of endothelial cell function is interaction with the extracellular matrix (ECM) proteins that bind growth factors and anchor cells to the vessel wall. Previously, we showed that basement membrane proteins modulate endothelial cell behaviors involved in wound healing and angiogenesis, and that estradiol augments endothelial cell-matrix interactions. The goal of this study was to determine the effect of ECM proteins and estradiol on HCAEC secretion of bFGF, a “wound healing” cytokine that mediates endothelial cell migration, proliferation, and other responses to vascular wall injury. Female HCAEC were grown to confluence in estrogendepleted medium for 48 hours, and replated for experimentation onto plastic culture dishes coated with gelatin, type I collagen, type IV collagen, fibronectin, or laminin. After anchoring for 8 hours, the cells were treated for 2 hours with estradiol, 1.5 ng/ml (5 nM). The medium was then replaced with serum-free medium. After 5 hours, the conditioned media and the cell layers were harvested separately and evaluated for bFGF content by ELISA and immunoblot analysis. Secretion of bFGF into conditioned media of estradiol-depleted cultures was enhanced by 3-, 5-, and 6- fold on laminin, fibronectin, or gelatin, respectively. When the cells were supplemented with estradiol, there was a further 2-fold or 6-fold increase in bFGF secretion upon HCAEC binding to fibronectin or type IV collagen, respectively. Estradiol did not increase bFGF secretion by cells cultured on plastic or type I collagen. Intracellular bFGF content, determined from cell lysates, did not change under any experimental conditions. Similar results were obtained by immunoblot analysis. These results show that (1) secretion of bFGF is enhanced specifically by matrix substrata, and (2) estradiol enhances bFGF secretion on certain substrata. The data demonstrate that interaction among cells, matrix and estrogen facilitates secretion of an autocrine factor that is important for endothelial cell activity in vascular healing. One mechanism by which estrogen exerts a cardioprotective effect may thus be stimulation of specific cytokine/growth factor secretion.