The present study was undertaken to investigate whether withdrawal of estrogen and progesterone (EP-withdrawal) stimulates prostaglandin F2α (PGF2α) production through oxygen radical (ROS)-induced NF-κB activation in human endometrial stromal cells (ESC). To study the EP-withdrawal, ESC that had been treated with estradiol (E, 10^-8 M) and medroxyprogesterone acetate (MPA, 10^-6 M) for 12 days were then incubated with or without E+MPA for a further 11 days. PGF2α concentrations in the medium and cyclooxygenase-2 (COX-2) mRNA levels were significantly increased after EP-withdrawal, while they were unchanged by the continuous treatment with E+MPA. When ESC were incubated with N-acetyl-L-cysteine (Nac, 50 mM), an antioxidant, during EP-withdrawal, Nac blocked the increases in PGF2α production and COX-2 mRNA expression caused by EP-withdrawal. Next, we examined whether ROS generated in response to EP-withdrawal acted through NF-κB activation. Electrophoretic mobility shift assay revealed that EP-withdrawal caused marked increases in NF-κB DNA binding activity, which was completely suppressed by Nac. Furthermore, when ESC were incubated with MG132 (3 μM), which inhibits NF-κB activation, during EP-withdrawal, MG132 blocked the increases in PGF2α production and COX-2 mRNA expression caused by EP-withdrawal. In conclusion, EP-withdrawal stimulates COX-2 expression and PGF2α production through ROS-induced NF-κB activation, suggesting a possible mechanism for menstruation.