Streptokinase is a plasminogen activator protein produced by several strains of β-hemolytic streptococci. Random mutagenesis of streptokinase was carried out for the determination of critical amino acid residues in plasminogen activation. We selected and sequenced 14 streptokinase mutants with no plasminogen activation activity on skim milk-plasminogen overlay plate. Specific activities of the selected streptokinase mutants were determined with chromogenic assay. Eight mutants (V19F, V35E, E85D, L292R, D325P, D341E, I345N, and M369L) resulted in greatly decreased amidolytic activities. However, unexpectedly, six mutants (D41C, S44K, S44P, R45P, H48T, and D220G) showed substantial amidolytic activities comparable to that of wild type. Moreover, five-point mutations were concentrated on the Asp41-His48 region. These data indicate that the Asp41-His48 region in a streptokinase-plasminogen binary complex plays an important role in binding to a substrate plasminogen.