Alpha2-antiplasmin (alpha2-AP) and plasminogen activator inhibitor-1 (PAI-1) are the main physiological inhibitors of the plasminogen/plasmin system in mammalian plasma. In the present study, the relative importance of both inhibitors was evaluated with the use of mice with single or combined deficiency of alpha2-AP and PAI-1 in the same genetic background. Mice with combined deficiency (alpha2-AP-/-:PAI-1-/-) are viable, develop normally and are fertile. After amputation of the tail, bleeding times are prolonged (>15 min) in alpha2-AP-/-: PAI-1-/- mice, as compared to double wild-type or single deficient mice (4.6 to 10 min). Spontaneous lysis after 4 h of intravenously injected 125I-fibrin labeled plasma clots is significantly higher in mice with alpha2-AP deficiency both in the PAI-1+/+ background (89+/-2% versus 42+/-3%; p = 0.002) and in the PAI-1-/- background (83+/-4% versus 53+/-5%; p = 0.002). PAI-1 deletion in the alpha2-AP+/+ or alpha2-AP-/- background, however, has no significant effect (p = 0.13 or 0.18, respectively). Four hours after endotoxin injection, fibrin deposition in the kidneys is not significantly affected by PAI-1 deletion in mice with alpha2-AP+/+ or alpha2-AP-/- background (p = 0.07 and 0.19, respectively). In contrast, alpha2-AP deletion causes significantly reduced fibrin deposition in the PAI-1+/+ background (p = 0.01). Endotoxin injection causes a dramatic increase in PAI-1 antigen levels in kidney extracts of PAI-1+/+ animals, without effect on alpha2-AP levels. Taken together, these data indicate that the higher endogenous fibrinolytic capacity observed in mice with combined deficiency is mainly due to the lack of alpha2-AP and suggest a less important role for PAI-1.