To investigate the mechanism of ultrasound-enhanced fibrinolysis, the effect of ultrasound on binding of tissue plasminogen activator (t--PA) to fibrin was examined because t--PA--fibrin interactions are important in determining the rate of fibrinolysis. A novel system to quantitate t--PA--fibrin interactions was developed in which radiolabeled, active site blocked t--PA flowed through a fibrin gel at constant rate and temperature, and specific binding was determined by monitoring incorporation of radiolabel. Exposure to 1-MHz ultrasound to 2 W cm[sup 2] resulted in no significant change in the kD of t--PA binding to either cross-linked or noncross-linked fibrin. Ultrasound, however, significantly increased the B[inf max], and the molar binding ratio increased in the presence of ultrasound from 1.85 (mol t--PA/mol fibrin) to 2.54 (mol t--PA/mol fibrin) for noncross-linked fibrin and from 1.19 (mol t--PA/mol fibrin) to 1.43 (mol t--PA/mol fibrin) for cross-linked fibrin. Ultrasound resulted in no significant changes in binding of t--PA to fibrin monomer in the same system. It is concluded that exposure to ultrasound increases binding of t--PA to polymerized fibrin, possibly by exposing additional binding sites in the polymer which are inaccessible in the absence of ultrasound.