Ultrasound (US) accelerates enzymatic fibrinolysis in vitro and in animal models, and may be a useful adjunctive therapy for clinical thrombolysis. Successful clinical application will depend on the selection of appropriate US parameters to optimize fibrinolytic enhancement while limiting adverse effects, including heating. Most studies have been done at megahertz frequencies, but tissue penetration is better and heating less at lower frequencies. We have, therefore, now investigated the effects of continuous-wave and pulsed US on fibrinolysis at midkilohertz frequencies. Fibrinolysis with tissue plasminogen activator (t-PA) was measured by solubilization of radiolabeled fibrin exposed to a calibrated US field in a temperature-controlled water bath. There was significant enhancement of fibrinolysis at frequencies of 27, 40 and 100 kHz, with the greatest effect observed at 27 kHz. The largest effect was observed with continuous-wave US, but significant acceleration was also observed with peak intensities of 1 W/cm(2) duty cycles of 10% and 1%. At a 10% duty cycle, there was approximately 60% of the fibrinolytic enhancement observed with continuous-wave exposure, indicating a clear advantage of pulsing to optimize fibrinolytic effect and limit exposure. We conclude that US in the range of 27 to 100 kHz is effective in accelerating fibrinolysis at intensities and pulsing conditions that minimize the probability of heating and cavitation in clinical applications.