The objective of this study was to investigate tumor necrosis factor α (TNF-α) expression, the presence of functional TNF-α receptors, and expression of TNF receptor type I (TNF-RI) mRNA in the bovine corpus luteum (CL) during different stages of the estrous cycle. Reverse transcription (RT)-polymerase chain reaction (PCR) showed no difference in TNF-α mRNA expression during the estrous cycle. Concentrations of TNF-α in the CL tissue increased significantly from the mid to the late luteal stage and decreased thereafter (P < 0.05). An RT-PCR analysis showed higher levels of TNF-RI mRNA in CL of Days 3–7 than of other stages (P < 0.05). ¹²⁵I-TNF-α binding to the membranes of bovine CL was maximal after incubation at 38°C for 48 h. The binding was much greater for TNF-α than for related peptides. A Scatchard analysis revealed the presence of a high-affinity binding site in the CL membranes collected at each phase of the estrous cycle (dissociation constant: 3.60 ± 0.58–5.79 ± 0.19 nM). In contrast to TNF-RI mRNA expression, the levels of receptor protein were similar at each stage of the estrous cycle. When cultured cells of all luteal stages were exposed to TNF-α (1–100 ng/ml), TNF-α stimulated prostaglandin F_2α and prostaglandin E₂ secretion by the cells in a dose-dependent fashion (P < 0.01), especially during the early luteal phase, although it did not affect progesterone secretion. These results indicate the local production of TNF-α and the presence of functional TNF-RI in bovine CL throughout the estrous cycle, and suggest that TNF-α plays some roles in regulating bovine CL function throughout the estrous cycle.