Thrombolysis with single and combined four-hour intravenous (IV) infusions of recombinant tissue-type plasminogen activator (rt-PA), recombinant single-chain urokinase-type plasminogen activator of 54,000 molecular weight (mol wt) (rscu-PA), and rscu-PA-32 kD, an rscu-PA derivative of 32,000 mol wt was studied in a femoral vein thrombosis model in the dog and in a jugular vein thrombosis model in the rabbit. In both species, the dose-response curves were linear, and no systemic activation of the fibrinolytic system or fibrinogen breakdown was observed. The steady-state levels of rt-PA-, rscu-PA-, and rscu-PA-32 kD-related antigens in plasma were proportional to the infusion rates. In the dog model, 25% lysis was obtained with 0.11 mg/kg rt-PA, 0.8 mg/kg rscu-PA, and 0.37 mg/kg rscu-PA-32 kD. Combinations of rt-PA and rscu-PA were 2.6 times more active (P less than .005) than anticipated on the basis of their pharmacologic additive effects, whereas combinations of rt-PA and rscu-PA-32 kD were 2.7 times more active (P less than .05). In the rabbit model, 25% lysis was obtained with 0.24 mg/kg rt-PA, 0.75 mg/kg rscu-PA, and 1.25 mg/kg rscu-PA-32 kD. Combinations of rt-PA and rscu-PA have a fivefold synergistic interaction, but surprisingly no synergism was observed between rt-PA and rscu-PA-32 kD. This study shows that synergism between rt-PA and rscu-PA occurs both in rabbits and dogs in a relatively narrow concentration range that allows a fractional reduction of the total equipotent dose by a factor of 2.5-fold to fivefold. Combination therapy is not associated with systemic fibrinolytic activation. This range of synergistic interaction, although limited, may be useful in devising the best thrombolytic therapy for patients with thromboembolic disease.