Luteal development and demise are characterized by substantial tissue destruction and remodeling, which is associated with local production of plasminogen activation. Recently we reported involvement of tissue-type plasminogen activator (tPA) in luteolysis in rhesus monkeys. In this study, we further investigated changes in expression of both tPA and urokinase-type plasminogen activator (uPA) activity during various developmental stages of rat corpus luteum (CL) of pregnancy and their possible physiological roles in luteolysis. Rat CL or dispersed luteal cells in vitro are capable of producing both tPA and uPA, and a plasminogen activator inhibitor type-1, in a stage-dependent manner. However, only tPA activity significantly increases in late phases of CL development. Furthermore, the increase in tPA activity in the CL is well correlated with a sharp decrease in luteal progesterone production. Addition of exogenous tPA to the luteal culture considerably decreases progesterone production. In contrast, immunoneutralization of endogenously produced tPA activity by inclusion of tPA monoclonal antibody in the culture results in a significant increase in luteal progesterone production. It is therefore suggested that tPA may also be involved in suppression of rat luteal function. This hypothesis is further supported by the findings that interferon-gamma significantly inhibits luteal basal and hCG-stimulated progesterone production and also stimulates basal and hCG-induced tPA activity. On the basis of the data provided in this study and similar findings in monkeys, we conclude that endogenously produced tPA in late phase of CL development may regulate luteal regression through local autocrine or paracrine action.