Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine produced by T cells and macrophages. A number of tissues also produce MIF during states of active differentiation and/or proliferation. The purpose of this study was to determine whether MIF is present in the corpus luteum (CL). The steady-state mRNA for MIF was examined in CL by Northern analysis on Day 5, Days 9–12, and Day 18 of the estrous cycle and at 0.5, 1, 4, 12, 24, and 36 h after a luteolytic injection of prostaglandin F_2α (PGF_2α) (n = 4 CL per time point). The greatest amount of MIF mRNA was observed in Day 5 CL compared with midcycle and Day 18 CL. Messenger RNA for MIF in CL collected 0.5 h post-PGF_2α was greater than in midcycle and all other regressing CL. Immunohistochemical analysis (n = 4) revealed that MIF was present in the bovine CL throughout the estrous cycle and appeared to be localized to large luteal cells. It was concluded that MIF is produced within the bovine CL, mRNA expression is maximal in the early CL, and the protein is primarily localized to large luteal cells. The functional significance of MIF remains to be determined.