Many studies suggest that the plasminogen activator (PA) system plays a role in the proteolytic degradation of the follicle wall at the time of ovulation. Consistently, the ovulation efficiency is reduced by 26% in mice where both physiological PA genes have been inactivated. To reveal the mechanism behind reduced ovulation efficiency in PA-deficient mice and its effect on ovarian proteolysis. we have studied the regulation of plasmin activity in the ovaries of 25-day-old wild-type mice and mice with deficient PA gene function during gonadotropin-induced ovulation. In wild-type mice the plasmin activity was low in ovarian extracts from mice treated with pregnant mare's serum gonadotropin. However, this activity was increased between 2-8 h after an ovulatory dose of human choriogonadotropins. In mice lacking either tissue-type PA (tPA) or PA inhibitor type 1 (PAI-1) the plasmin activity levels prior to ovulation were similar to wild-type mice, while extracts prepared from urokinase-type PA (uPA) deficient mice had 10% or less of the plasmin activity. This indicates that most of the plasmin activity in the mouse ovary is generated by uPA. In addition, as the ovulation efficiency is impaired in tPA/uPA-deficient mice but appears normal in uPA-deficient mice, our data indicates that the amount of plasmin generated by PAs prior to ovulation in wild-type mice greatly exceeds the amount required for efficient ovulation.