The effects of both clot-bound and circulating plasminogen activator inhibitor-1 (PAI-1) on endogenous fibrinolysis were investigated in a rat model of pulmonary embolism. Iodine-125 fibrin(ogen)-labeled blood-clot homogenates were delivered through the left jugular vein to the lung microvasculature, and the subsequent extent of the clot lysis was monitored by measuring the release of 125I-fibrin degradation products (FDPs) into the blood. Clots that had incorporated activated PAI-1 ex vivo were subsequently protected from dissolution in vivo in a dose-responsive manner (half-maximal concentration [IC50] = 4.3 micrograms/ml). PAI-1-containing clots also resisted lysis, as measured by the release of the specific FDP D-dimer. Plasma levels of plasminogen activator (PA) and PAI activity were unaltered by administration of PAI-1-containing clots, and the clot-protective effects of clot-bound PAI-1 were reversed by exogenous tissue-type plasminogen activator administration. Clot lysis was also inhibited in a dose-responsive manner (IC50 = 58 micrograms/kg) by intravenous bolus delivery of activated PAI-1 to the circulation. The clot-protective effects of circulating PAI-1 were correlated with dose-dependent increases in plasma PAI-1 antigen and activity levels and decreases in plasma PA levels (IC50 = 37 micrograms/ml). There was no evidence of any accumulation of circulating PAI-1 in the lungs. Latent PAI-1, whether delivered with or delivered after the clot homogenates, did not affect the clot-lytic process. Activated and latent PAI-1 was cleared from the circulation in a monophasic manner, with a half-life of approximately 32 and 7 minutes, respectively. The results indicate that both clot-bound and circulating PAI-1 are potent inhibitors of fibrinolysis in vivo. Clot-bound PAI-1 may inhibit PAs in the immediate vicinity of the clots, whereas circulating PAI-1 may act systemically by controlling overall levels of PAs present in the blood.