We examined whether prostaglandins (PGs) and nitric oxide (NO) mediate tumor necrosis factor (TNF) actions in the estrus cycle. On Day 14 of the cycle, the following solutions were infused into the aorta abdominalis of a total of 51 heifers (Experiments 1 and 2): saline; 1 or 10 μg of TNF; 480 mg indomethacin (INDO), an inhibitor of prostaglandin H synthase; 800 mg L-NAME, an inhibitor of NO synthase; and TNF (1 or 10 μg) in combination with INDO or L-NAME. TNF at 1 μg infused directly into aorta abdominalis increased the level of PGF_2alpha and decreased the level of progesterone (P4) in the peripheral blood and shortened the estrus cycle. The high TNF dose stimulated P4 and PGE₂ and prolonged the corpus luteum (CL) lifespan. INDO blocked the effects of both TNF doses on the CL lifespan and hormone output. L-NAME completely blocked the effects of the luteolytic TNF dose, whereas the effects of the luteotropic TNF dose were not inhibited. In Experiment 3 (Day 14), saline or different TNF doses were infused into the jugular vein (n = 9) or into the uterine lumen (n = 18). The CL lifespans of the different groups were not different when TNF was infused into the jugular vein. Although high TNF doses (1 and 10 μg) infused into the uterine lumen prolonged the CL lifespan, low doses (0.01 and 0.1 μg) induced premature luteolysis. We suggest that the actions of exogenous TNF on the CL lifespan depend on PG synthesis stimulated by TNF in the uterus. TNF at low concentrations initiates a positive cascade between uterine PGF_2alpha and various luteolytic factors, including NO, to complete premature luteolysis. PGE₂ is a good candidate mediator of the luteotropic actions of exogenous TNF action.