A number of hybrid plasminogen activator genes were constructed from the t-PA and u-PA cDNAs and expressed using a bovine papilloma virus vector and mouse C-127 cells. Hybrid A was constructed by replacing the finger (F) and EGF domains of t-PA with the EGF and Ku domains of u-PA, while hybrids B and C had an extra Ku inserted before or after the double kringle (K1-K2) region of t-PA respectively. While all the hybrids showed comparable enzymatic activities towards a small substrate (S-2288), they had different activities in binding to fibrin clots as well in the fibrin-dependent plasminogen activation, the order of activities being: t-PA greater than or equal to hybrid B greater than hybrid C greater than hybrid A. Carbohydrate analysis showed that while hybrid C, like rt-PA, had at least one high-mannose type sugar chain (probably at residue 117 in K1), the other hybrids had only complex-type carbohydrates suggesting that domain interaction in t-PA might influence glycan processing. Pharmacokinetic studies in dog showed that hybrid B had a significantly longer plasma half-life than rt-PA. Thrombolytic efficacies of hybrid B and rt-PA were compared in dog model using an artificially induced coronary thrombus. Complete thrombolysis was achieved with 18 mg and 50 mg dosages for hybrid B and rt-PA respectively. These data show the superior pharmacokinetic and thrombolytic properties of hybrid B compared to rt-PA.