The experiments reported here were carried out to define in greater detail actin's stimulation of plasmin generation by t-PA. Actin did not alter t-PA's hydrolysis of a synthetic substrate, and thus is unlikely to have a direct effect upon t-PA's proteolytic activity. When studied in a single-stage assay, actin accelerated t-PA-mediated plasmin generation from both Glu-plasminogen and Lys-plasminogen, indicating the central role of ternary complex formation. Although actin does not appear to bind two-chain urokinase (tcu-PA), it stimulates tcu-PA's cleavage of Glu-plasminogen. This finding suggests that actin alters the conformation of Glu-plasminogen to an open form. The failure of actin to increased plasmin generation by tcu-PA acting on Lys-plasminogen, which is in an open configuration, is consistent with this interpretation. Immunoglobin G, which shares with actin the property of binding to Glu-plasminogen after nicking by plasmin, did not stimulate tcu-PA's cleavage of Glu-plasminogen, indicating the uniqueness of actin's effects and suggesting interactions between actin and plasminogen at multiple binding sites. Unlike fibrin and heparin, whose stimulation of t-PA is related to polymer length actin is able to stimulate t-PA when presented in either a monomeric or polymeric form. Denaturation of actin by exposure to urea and guanidine increased its ability to stimulate plasmin generation by t-PA. Because actin's structure is maintained by a noncovalently bound adenine nucleotide (ATP or ADP), exposure to ATP/ADPases found in plasma and on cell membranes might also result in its denaturation. Actin treated with an enzyme functionally similar to such ecto-ATP/ADPases, potato apyrase, was more potent than native actin in stimulating plasmin generation by t-PA. The effects of apyrase were blocked by the addition of the plasma actin-binding proteins, gelsolin and the vitamin D-binding protein (DBP). Thus, denaturation of actin may occur in under physiologic conditions, with potential biological consequences. Actin thus appears to be unique with regard to its interactions with the fibrinolytic system and plasma actin-binding proteins may serve to protect the host from the effects of denatured actin.