Exposure to 2-MHz transcranial diagnostic ultrasound enhances the thrombolytic activity of intravenously administered tissue plasminogen activator (IV-tPA) in acute ischemic stroke (sonothrombolysis). However, rates of arterial recanalization vary widely, depending upon the clot burden, its location, and stroke subtype. We evaluated the influence of age and cholesterol level of the blood clots on sonothrombolysis in an in vitro model. To "age" the clots, serum was replaced by fresh blood periodically. We increased the cholesterol content of the clots by adding cholesterin to the blood. The clots were lysed by tPA and/or transcranial Doppler ultrasound sonication for 1 h. The extent of thrombolysis induced by various treatment protocols (controls, sonication, tPA, and sonothrombolysis) was evaluated with relative changes in the clot weights and in the clot structure by scanning electron microscopy (SEM) at end of the experiment. Sonothrombolysis induced significantly higher weight reduction in fresh clots (37.3 % in 2-h old clots versus 24.8 % in 10-h ones, p < 0.005) as well as the clots with higher cholesterol levels (41.7 versus 30.6 % in normal cholesterol clots, p < 0.005). SEM demonstrated patterns of clot dissolution among various treatment modalities. Sonothrombolysis induced better clot lysis in fresh thrombi with high cholesterol levels.