Abstract Fibrinolysis is accelerated in vitro in an ultrasound field, and externally applied high frequency ultrasound also accelerates thrombolysis in animal models. Although the mechanism of this effect is not known, ultrasound does not cause mechanical disruption of clots but rather accelerates enzymatic fibrinolysis. To determine if accelerated fibrinolysis could be related to increased transport of enzyme into clot, we have examined the effect of insonification on the distribution of plasminogen activator between clot and surrounding fluid in vitro. Plasma clots were overlayed with plasma containing 125I-radiolabeled, activesite-blocked recombinant tissue plasminogen activator (rt-PA) and incubated in the presence of 1-MHz ultrasound at 4 W/cm 2 or in the absence of ultrasound. The rate of uptake of rt-PA was significantly faster in the presence of ultrasound, reaching 15.5 ± 1.4% at 4 h compared to 8.2 ± 1.0% in the absence of ultrasound ( p < 0.0001). Similarly, ultrasound increased transport of enzyme from the clot into the surrounding fluid. To determine the effect of ultrasound on the spatial distribution of enzyme, plasma clots were overlayed with plasma containing radiolabeled rt-PA and incubated in the presence or absence of ultrasound. The clots were then snap-frozen, and the radioactivity in serial cryotome sections was determined. Exposure to ultrasound altered the rt-PA distribution, resulting in significantly deeper penetration of rt-PA into the clots. We conclude that exposure to ultrasound increases uptake of rt-PA into clots and also results in deeper penetration. These effects of ultrasound on enzyme transport may contribute to the accelerated fibrinolysis observed in an ultrasound field.