Streptokinase (SK) is an extracellular protein produced by many strains of hemolytic streptococci, which lyses blood clots by activating plasminogen to poasmin. The mechanism of poasminogen activation by streptokinase had long been investigated, however, there are little reports about the three dimensional structure of streptokinase and its interaction with plasminogen in detail. Recently, it was reported that C-terminal amino acids of streptokinase may have potential importane in structural and functional aspects. To elucidate the structural and functional roles of C-terminal amino acids of streptokinase, several C-terminal deleted streptokinase genes were constructed by nested deletion of streptokinase coding gene with exonuclease III. The C-terminal deleted streptokinases were expressed in E. coli and purified by affinity chromatography using plasminoge-coupled Sepharose 4B. DSK33 (mutant SK lost 33 amino acids from the C-termunus of a native SK) and DSK40 behaved like as a native SK in the column. On the other hand, DSK46 showed a smaller peak than a native streptokinase and DSKs lost 47 residues or more did not bind to the column. CLN hydrolsys test of purified DSKs showed that 40 residues deletion from the C-terminus dis not affect the activation activity. However, deletion of 46 or more amino acids resulted in dramatically reduced plasminogen activation activity. These results propose that residues 40 to 47 from C-terminus consist in a binding site of streptokinase with plasminogen.