Abnormal uterine bleeding is the leading indication for discontinuation of long-term progestin-only contraceptives (LTPOCs). Histological sections of endometria from LTPOC-treated patients display abnormally enlarged blood vessels at bleeding sites. Paradoxically, a trend toward reduced endometrial perfusion in LTPOC users has been reported in these patients. We hypothesized that hypoxia/reperfusion-induced free radical production inhibits the expression of angiopoietin-1 (Ang-1), a vessel stabilizing factor, leaving unopposed the effects of endothelial Ang-2, a vessel-branching and permeability factor. Immunohistochemical studies confirmed selective decreases in stromal cell Ang-1 in LTPOC-exposed endometrium. To indirectly assess whether LTPOC enhances endometrial free radical production, immunostaining was conducted for the phosphorylated form of the stress-activated kinases SAPK/JNK and p38. These kinases were greatly increased in endometria from LTPOC-treated patients. Interestingly, the endothelial cells but not the stromal cells displayed enhanced immunostaining for the phosphorylated mitogen-activated kinase (pMAPK) after LTPOC treatment. To further examine the effects of progestin, hypoxia, and reactive oxygen species (ROS) on the regulation of Ang-1 and Ang-2 as well as the activation of MAPK, SAPK/JNK, and p38 by the relevant cell types, we conducted in vitro studies with cultured human endometrial stromal cells (HESCs) and human endometrial endothelial cells (HEECs). Cultures of HESCs were treated with vehicle control, estradiol (E2), or with medroxyprogesterone acetate ± E2 under hypoxic and normoxic conditions. Although medroxyprogesterone acetate but not E2 increased Ang-1 expression, hypoxia greatly decreased Ang-1 protein and mRNA expression. In contrast, HESCs did not appear to express Ang-2 protein or mRNA. Conversely, cultured HEECs did not appear to express Ang-1, but expressed Ang-2, the levels of which were significantly increased by hypoxia. Hypoxia also induced the phosphorylation of SAPK/JNK and p38 in both cultured HESCs and HEECs. Moreover, ROS such as that observed after hypoxia/reperfusion resulted in the activation of SAPK/JNK and p38 in HESCs and HEECs and inhibited Ang-1 in cultured HESCs. These effects could be blocked by oxygen radical scavengers. Consistent with the in vivo studies, MAPK was activated after ROS treatment in HEECs but not in HESCs. Our findings suggest that LTPOC-induced endometrial bleeding occurs as a result of hypoxia/reperfusion-induced free radicals that directly damage vessels and alter the balance of Ang-1 and Ang-2 to produce the characteristic enlarged and permeable vessels that are prone to bleeding.