Endometriosis, one of the most frequently occurring gynecological disorders, is estrogen dependent and is often associated with immunological changes. These include increased macrophage activation and infiltration into the endometriotic implants themselves as well as the peritoneal cavity where the implants often develop. Despite the critical role estrogens play in the development of endometriosis, the biochemical mechanisms of their action remain unclear. In the present study we report that estradiol (E2) enhances endometriotic cell responsiveness to the proinflammatory cytokine interleukin-1beta by up-regulating interleukin-1-induced monocyte chemotactic protein-1 (MCP-1) expression at the level of both protein secretion and messenger ribonucleic acid (mRNA) synthesis, whereas progesterone had no significant effects. According to mRNA half-life experiments, E2 action does not seem to be due to increased MCP-1 mRNA stability but, rather, to a higher level of transcription, as shown by run-on analysis. Interestingly, immunohistochemical analysis of MCP-1 expression in endometriotic tissue showed intense immunostaining in both epithelial glands and stroma regardless of the menstrual cycle phase, which is consistent with the cell culture data and indicates that MCP-1 expression is not subject to cyclic variation. The findings of the present study for the first time provide evidence that E2 up-regulates, although in an indirect way, the expression of a potent chemotactic and activating factor by ectopic endometrial cells, which may occur locally in the inflammatory site and contribute to peritoneal macrophage recruitment and activation, and reveal a new means of E2 action in the pathophysiology of endometriosis.