To determine the physiological significance of tumor necrosis factor α (TNFα) in the regulation of luteolytic prostaglandin (PG) F_2α release by the bovine endometrium, the effect of TNF-α on PGF_2α output by the endometrial tissues in vitro was investigated and compared with the effect of oxytocin (OT). Furthermore, the presence of specific receptors for TNFα in the bovine endometrium during the estrous cycle was determined. Endometrial slices (20–30 mg) taken from six stages of the estrous cycle (estrus: Day 0; early I: Days 2–3; early II: Days 5–6; mid-: Days 8–12; late: Days 15–17; and follicular: Days 19–21), as determined by macroscopic examination of the ovaries and uterus, were exposed to TNFα (0.06–6 nM) and/or OT (100 nM). OT stimulated PGF_2α output at the follicular stage and at estrus (P < 0.001), but not at the late luteal stage. On the other hand, the stimulatory effects of TNFα on PGF_2α output were observed not only at the follicular stage but also at the late luteal stage (P < 0.001). When the endometrial tissues at late luteal stage were simultaneously exposed to TNFα (0.6 nM) and OT (100 nM), the stimulatory effect on PGF_2α output was higher than the effect of TNFα or OT alone (P < 0.05). Specific binding of TNFα to the bovine endometrial membranes was observed throughout the estrous cycle. The concentration of TNF-α receptor at the early I luteal stage was less than the concentrations at other luteal stages (P < 0.01). The dissociation constant (K_d) values of the endometrial membranes were constant during the estrous cycle. The overall results lead us to hypothesize that TNFα may be a trigger for the output of PGF_2α by the endometrium at the initiation of luteolysis in cattle.