The present in vitro study investigated dose-, time- and temperature-dependent effects of two-chain urokinase plasminogen activator (u-PA, urokinase) on normal citrated plasma. When 10 micrograms/ml u-PA were added to pooled normal plasma and incubated for 30 min at an ambient temperature (25 degrees C), alpha 2-antiplasmin decreased to 8% of the control value. Incubation on ice yielded a decrease to 45% of control, whereas alpha 2-antiplasmin was fully consumed at 37 degrees C. Fibrinogen and plasminogen fell to 46% and 39%, respectively, after a 30 min incubation at 25 degrees C. Thrombin time prolonged to 190% of control. Various inhibitors were studied with respect to their suitability and efficacy to prevent these in vitro effects. Aprotinin exhibited a good protective effect on fibrinogen at concentrations exceeding 500 KIU/ml plasma. Its use, however, was limited due to interferences with some haemostatic assays. We could demonstrate that L-Glutamyl-L-Glycyl-L-Arginyl chloromethyl ketone (GGACK) and a specific polyclonal anti-u-PA-antibody (anti-u-PA-IgG) effectively inhibited urokinase-induced plasmin generation without interfering with haemostatic assays. The anti-u-PA-antibody afforded full protection of alpha 2-antiplasmin at therapeutic levels of u-PA. It is concluded that u-PA in plasma samples from patients during thrombolytic therapy may induce in vitro effects which should be prevented by the use of a suitable inhibitor such as GGACK or specific anti-u-PA-antibody.