Recombinant tissue-type plasminogen activator (rt-PA), produced by expression of the genomic t-PA DNA from the JMI-229 cell line, which is of rat origin, in the host cell line, was purified to homogeneity. JMI-229 rt-PA was obtained essentially as a single chain molecule which was quantitatively converted to a two-chain moiety by treatment with plasmin. The plasminogen activating potential of single chain JMI-229 rt-PA was 5-fold lower than that of commercially available human rt-PA (Actilyse) in the absence of fibrin, but comparable in the presence of fibrin; it showed a concentration-dependent binding to fibrin, with a significantly more pronounced binding than Actilyse at low fibrin concentration (85 +/- 8% versus 20 +/- 7% at 0.025 mg/ml fibrin; p = 0.004). In human plasma in the absence of fibrin, the concentrations of both single chain and two-chain JMI-229 rt-PA required to induce 50% fibrinogen degradation in 2 h, were about 15-fold higher than those of Actilyse. Both single chain and two-chain forms of JMI-229 rt-PA and of Actilyse induced a similar time- and concentration-dependent lysis of a 125I-fibrin-labeled plasma clot immersed in human plasma, in the absence of significant systemic fibrinolytic activation. Equally effective concentrations (causing 50% clot lysis in 2 h) were 0.11 or 0.10 micrograms/ml for single chain or two-chain JMI-229 rt-PA, as compared to 0.11 or 0.15 micrograms/ml for single chain or two-chain Actilyse.(ABSTRACT TRUNCATED AT 250 WORDS)