The specific thrombolytic properties of urokinase and three molecular forms of single-chain urokinase-type plasminogen activator (scu-PA) were compared in a human plasma milieu in vitro and in an experimental thrombosis model in rabbits. These scu-PA molecules included Mr 54,000 scu-PA from human urine (urinary scu-PA), scu-PA from conditioned media of a human lung adenocarcinoma cell line (CALU-3,ATCC,HTB-55) (cellular scu-PA) and an Mr 32,000 proteolytic derivative of cellular scu-PA (scu-PA-32k). All four molecular forms induced significant lysis of a 125I-labeled human plasma clot immersed in citrated human plasma at concentrations between 50 and 200 IU/mL. None of the four showed absolute fibrin-specificity, but at equivalent lytic dose the three single-chain forms appeared to cause less fibrinogen degradation and alpha 2-antiplasmin consumption than two-chain urokinase. In addition, the fibrinolytic potential of the three single-chain forms was largely maintained during pre-incubation in plasma for up to 48 hours whereas that of urokinase was completely inhibited. Intravenous (IV) infusion of cellular scu-PA or scu-PA-32k into rabbits with a 125I-labeled thrombus in the jugular vein caused significant dose-dependent lysis at concentrations ranging from 8,700 to 35,000 and from 9,000 to 36,000 IU/kg respectively. Clot lysis was accompanied by minor alpha 2-antiplasmin consumption or fibrinogen breakdown. In contrast, urokinase induced lysis at doses between 20,000 and 200,000 IU/kg, but at higher doses was associated with significant systemic activation of the fibrinolytic system. It is concluded that scu-PA obtained from CALU-3 cell cultures has identical thrombolytic properties to that obtained from urine. In addition, the scu-PA-32k proteolytic derivative has the same fibrin-specific thrombolytic properties as the intact molecule. Cellular scu-PA and scu-PA-32k may therefore constitute more readily available alternatives for clot-selective thrombolytic therapy in man.