Streptokinase, one of the major blood-clot-dissolving agents, catalyzes the conversion of the plasma-zymogen (plasminogen) to the serine protease plasmin. Streptokinase genes fromtwo different S. equisimilis strains, isolated from equine and human, were cloned and expressed in Escherichia coli. Cloned streptokinase genes were investigated for their expression in E. coli by Western blot analysis and radial caseinolysis assay. Recombinant genes of streptokinase genes were similar to their native counterparts in terms of their molecular masses and exhibited preferential plasminogen activity. PAGE and Radial caseinolysis studies demonstrated that streptokinases secreted by streptococci from different hosts were able to differentially activate only the plasminogen derived fromthe same host. Streptokinase-plasminogen interaction indicated that human and equine plasminogens were cleaved at the same highly conserved site and an altered form of streptokinase was produced. This work strongly supports the idea that these streptokinases must share a common plasminogen binding domain and species-specific activation of plasminogen by streptokinases which reflecting the origin of the streptococcal isolate and the role of the streptokinase in the pathogenesis of streptococcal infections.