Three t-PA mutants, t-PA del K1 (with deletion of K1 domain), t-PA del (296-302) (with deletion of PAI-1 binding site) and their combination mutant t-PA del (K1, 296-302), were constructed by DNA recombination and site-directed mutagenesis techniques. Then the three t-PA mutants were transiently expressed in COS-7 cells, and in addition, the combination mutant t-PA del (K1, 296-302) was stably expressed in CHO cells. The biological analysis of the expression products demonstrated that t-PA del (296-302) and t-PA del (K1, 296-302) had obtained the resistance to inhibition by PAI-1. In addition, the half-life of t-PA del (K1, 296-302) in rat plasma was increased 6 fold while the mutant affinity for fibrin was only slightly affected. Therefore, it was reasonable to consider that the mutant t-PA del (K1, 296-302) may become a potent candidate of new thrombolytic agent.