The turnover of recombinant pro-urokinase (Rec-pro-UK), recombinant urokinase (Rec-UK) and natural urinary urokinase (Nat-UK) was studied in rabbits and in squirrel monkeys (Saimiri sciureus). Following intravenous injection, urokinase activity disappeared rapidly from the blood. The initial disappearance rate could be described by a single exponential term with a t 1/2 of 3 to 6 min for each molecular form of urokinase in both species. Urokinase related antigen, measured with a radioimmunoassay in the plasma of the squirrel monkeys disappeared with a t 1/2 of 3.5 min for Rec-pro-UK, 6.0 min for Rec-UK and 8.0 min for Nat-UK. The clearance and organ distribution of Rec-pro-UK, Rec-UK and Nat-UK was studied with the use of 125I-labeled preparations. In each case the radioactivity initially disappeared rapidly from the plasma, also with a t 1/2 of a few min, but then the disappearance rate slowed down. Labeled Rec-UK in which the active site histidine was irreversibly blocked by alkylation, disappeared equally rapidly from the plasma. Measurement of the organ distribution of 125I at different time intervals revealed that all three types of urokinase were rapidly accumulated in the liver, which was followed by release of degradation products in the blood. Experimental hepatectomy prolonged the t 1/2 of each type of urokinase very markedly (t 1/2 greater than 30 min). These findings indicate that urokinase is rapidly removed from the blood by clearance and degradation in the liver. Recognition by the liver does not require a functional active site and is not mediated via carbohydrate side chains. Inactivation by plasma protease inhibitors does not represent a significant pathway of urokinase inhibition in vivo.