Menstruation is a complex process dependent on premenstrual release of inflammatory mediators and proteolytic enzymes from endometrial cells. Endometrial leukocytes are traditionally considered to be the major source of the inflammatory factors. However, evidence is emerging to suggest a role for decidualized endometrial stromal cells in the premenstrual inflammatory cascade. We sought to determine if withdrawal of hormone support (estrogen and progesterone) from decidualized endometrial stromal cells, in a model mimicking the precise timing leading to menstruation, activated inflammatory signaling pathways and downstream release of inflammatory mediators. Human endometrial stromal cells decidualized gradually over 12 days of estradiol and progestin treatment as evidenced by an increase in prolactin secretion. Withdrawal of hormone support from decidualized stromal cells resulted in a decrease in cytoplasmic IkappaB and a progressive increase in nuclear accumulation of NF-kappaB, as demonstrated by Western immunoblot and immunocytochemical analyses. Concomitant with nuclear translocation of NF-kappaB, hormone withdrawal led to production of a host of inflammatory mediators by the decidualized stromal cells, including IFN-alpha, IL-6, CCL11, GM-CSF, CCL2, IL1-RA, CXCL10, CXCL8, IL-12, IL-15, VEGF, and CCL5. Elevation of inflammatory mediators was not observed, however, upon hormone withdrawal in cells treated with the NF-kappaB inhibitor BAY 11–7085. Decidualized stromal cells are likely highly sensitive sensors of changing hormone levels. This provides a mechanism by which decidualized stromal cells may recruit inflammatory leukocytes into the premenstrual endometrium and contribute to the intense inflammation underlying this unique physiological process.