The effect of ultrasound on the rate of fibrinolysis has been investigated using an in vitro system. Plasma or blood clots containing a trace label of 125I fibrin were suspended in plasma containing plasminogen activator and intermittently exposed to continuous wave 1-MHz ultrasound at intensities up to 8 W/cm2. Plasma clot lysis at 1 h with 1 μg/ml recombinant tissue plasminogen activator (rt-PA) was 12.8±1.2% without ultrasound and was significantly (P = 0.0001) increased by exposure to ultrasound with greater lysis at 1 W/cm2 (18.0 ± 1.4%), 2 W/cm2 (19.3 ± 0.7%), 4 W/cm2 (22.8 ± 1.8%), and 8 W/cm2 (58.7 ± 7.1%). Significant increases in lysis were also seen with urokinase at ultrasound intensities of 2 W/cm2 and above. Exposure of clots to ultrasound in the absence of plasminogen activator did not increase lysis. Ultrasound exposure resulted in a marked reduction in the rt- PA concentration required to achieve an equivalent degree of lysis to that seen without ultrasound. For example, 15% lysis occurred in 1 h at 1 μg/ml rt-PA without ultrasound or with 0.2 μg/ml with ultrasound, a five-fold reduction in concentration. Ultrasound at 1 W/cm2 and above also potentiated lysis of retracted whole blood clots mediated by rt-PA or urokinase. The maximum temperature increase of plasma clots exposed to 4 W/cm2 ultrasound was only 1.7°C, which could not explain the enhancement of fibrinolysis. Ultrasound exposure did not cause mechanical fragmentation of the clot into sedimentable fragments, nor did it alter the sizes of plasmic derivatives as demonstrated by SDS polyacrylamide gel electrophoresis. We conclude that ultrasound at 1 MHz potentiates enzymatic fibrinolysis by a nonthermal mechanism at energies that can potentially be applied and tolerated in vivo to accelerate therapeutic fibrinolysis.