The objectives of this study were to determine whether tumor necrosis factor alpha (TNF alpha) is present in porcine corpus luteum (CL) and, if so, to ascertain which cells of the CL were associated with TNF alpha. The influence of the reproductive state of the animals on the amount or cellular localization of TNF alpha in the CL was also investigated. Western blot analysis demonstrated the presence of a 34-kDa immunoreactive TNF alpha-like protein in CL from nonpregnant and pregnant pigs. The intensity of TNF alpha immunostaining in bands observed in Western blots did not differ when luteal protein from nonpregnant pigs or from pregnant pigs was analyzed. Positive immunostaining for TNF alpha, determined by immunocytochemistry, was localized within endothelial cells of luteal sections. Positive immunocytochemical staining was recorded in all reproductive states examined except in CL that had undergone functional luteolysis (reduced progesterone concentration). A single-cell assay for TNF alpha secretion (a reverse hemolytic plaque assay) confirmed in vitro secretion of TNF alpha by a cell type in luteal tissue with a size and nuclear morphology similar to an endothelial cell. The presence of TNF alpha in the endothelial cells in functional CL is consistent with a role for TNF alpha in the regulation of vascular development of the CL in swine.