Tumor necrosis factor-alpha (TNFα) is a pleiotropic cytokine that has been implicated in apoptosis of many cell systems. However, the signal transduction of TNFα during the structural and functional regression of the corpus luteum (CL) is largely unknown. In this study, we investigate the role of TNFα in rat CL apoptosis and the involvement of monocyte chemoattractant protein-1 (MCP-1) and the modulating effect of the caspases in this process. An in vivo study of CL during pregnancy and postpartum using immunohistochemistry and Western blot analysis indicated that increases in TNFα correspond with luteal apoptosis approaching term (Day 22) and at postpartum (Day 3). CL apoptosis was further investigated using a whole-CL culture model of tropic withdrawal. An increase was observed in both low molecular weight (MW) DNA fragmentation and TUNEL staining from 0 h to 8 h in culture. CL apoptosis in vitro was associated with increased protein expression of both TNFα and MCP-1 as measured by immunohistochemistry and Western blot analysis. Using a whole-CL culture model, apoptosis was induced in vitro by TNFα as demonstrated by a dose-dependent increase in DNA fragmentation. Treatment of luteal cells with TNFα and both specific caspase inhibitors (Z-DEVD-FMK, Z-VEID-FMK, Z-IETD-FMK) or a general caspase inhibitor (Boc-D-FMK) prevented the effect of TNFα. CL regression involves the apoptotic deletion of luteal cells; the results of this study suggest that TNFα is possibly involved in this process. The observed increases in MCP-1 expression suggest the coordination of TNFα expression with the infiltration and activation of macrophages. Furthermore, the results demonstrate the importance of the caspases in the TNFα signal transduction pathway and suggest a hierarchy within the caspase family.