Tumor necrosis factor (TNF) α is an important physiological mediator of cell-to-cell communication. Recent observations suggest that TNFα is involved in the control of reproductive functions. The present study examined the role of TNFα in the secretion of factors involved in regulating smooth muscle contraction, such as prostaglandin E₂ (PGE₂), prostaglandin F_2α (PGF_2α), endothelin-1 (ET-1), and angiotensin II (Ang II), as it was in the original by the cow oviduct at different stages of the estrous cycle using an in vitro microdialysis system. Expression of mRNA for TNFα and its receptors (TNFα-R) was also evaluated. For microdialysis, the lumen of a portion (length, 10 cm) of the each oviductal segment was implanted with a dialysis capillary membrane, and TNFα (100 ng/ml) was infused for 4–8 h during a 16-h incubation period. The microdialysis system maintains cell-to-cell integrity and cell-to-cell communication, and it enables real-time observation of physiological changes in the luminal release of different substances. Concentrations of PG, ET-1, and Ang II in 4-h fractions were measured using second-antibody enzyme immunoassays. Infusion of TNFα stimulated oviductal secretion of PG, ET-1, and Ang II during the follicular and postovulatory stages, but not during the luteal stage. Expression of TNFα, TNFα-R type I, and TNFα-R type II mRNA was detected in the bovine oviduct by reverse transcription-polymerase chain reaction. High expression of both TNFαR types and ligands was detected during the follicular and postovulatory stages, whereas low expression was detected during the luteal stage. The results of the present study provide, to our knowledge, the first direct evidence that TNFα stimulates PG, ET-1, and Ang II secretion and that up-regulation of the TNFα system occurs in the cow oviduct during the periovulatory period. In conclusion, the TNFα system may optimize the release of contraction-related substances and modulate local contraction to regulate the oviductal transport of the gametes and embryo.