The following mutants of human tissue-type plasminogen activator (t-PA) were constructed by deletion mutagenesis of t-PA cDNA, expressed in Chinese hamster ovary cells and purified to homogeneity: (a) t-PA-delta FE:t-PA lacking both the fibronectin fingerlike (F) domain and the epidermal growth factor (E) domain, (b) t-PA-delta FE1X:t-PA-delta FE with the glycosylated 117Asn mutagenized to Gln, and (c) t-PA-delta FE3X:t-PA-delta FE with the three known glycosylated Asn residues replaced by Gln. The mutant and natural t-PA (Mel-t-PA obtained from melanoma cell culture) were infused intravenously for four hours into rabbits with jugular vein thrombosis at doses ranging between 0.12 and 0.75 mg/kg. Fifty percent thrombolysis, determined by interpolation, was obtained with 0.4 mg/kg Mel-t-PA, 0.37 mg/kg t-PA-delta FE, 0.2 mg/kg t-PA-delta FE1X, and 0.40 mg/kg t-PA-delta FE3X. These infusion rates resulted in plateau levels of t-PA antigen in plasma of 0.055, 2.1, 0.6, and 0.5 micrograms/mL, respectively. At 50% lysis, the residual fibrinogen 30 minutes after the end of the infusion was 100%, 81%, 100% and 85% of baseline, and the residual alpha 2-antiplasmin was 82%, 55%, 85%, and 90%, respectively. These results indicate that t-PA- delta FE1X and t-PA-delta FE3X have a specific thrombolytic activity and fibrin specificity comparable to that of Mel-t-PA. t-PA-delta FE has a comparable specific thrombolytic activity but a lower fibrin specificity than Mel-t-PA. After the end of the infusion, t-PA-related antigen disappeared from plasma with an initial t1/2 of four minutes for Mel-t-PA, 25 minutes for t-PA-delta FE, 42 minutes for t-PA-delta FE1X, and 14 minutes for t-PA-delta FE3X. It is concluded that t-PA can be modified by deletion mutagenesis to yield variants with a markedly longer half-life in the blood. Some of these variants have a specific thrombolytic activity and fibrin specificity similar to that of natural t-PA. These variants may be useful to identify the structures in t-PA responsible for its clearance, specific thrombolytic activity, and fibrin specificity in vivo.