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Streptokinase (SK) conformationally activates the central zymogen of the fibrinolytic system, plasminogen (Pg). The SK.Pg* catalytic complex binds Pg as a specific substrate and cleaves it into plasmin (Pm), which binds SK to form the SK.Pm complex that propagates Pm generation. Catalytic complex formation is dependent on lysine-binding site (LBS) interactions between a Pg/Pm kringle and the SK COOH-terminal Lys(414). Pg substrate recognition is also LBS-dependent, but the kringle and SK structural element(s) responsible have not been identified. SK mutants lacking Lys(414) with Ala substitutions of charged residues in the SK beta-domain 250-loop were evaluated in kinetic studies that resolved conformational and proteolytic Pg activation. Activation of [Lys]Pg and mini-Pg (containing only kringle 5 of Pg) by SK with Ala substitutions of Arg(253), Lys(256), and Lys(257) showed decreases in the bimolecular rate constant for Pm generation, with nearly total inhibition for the SK Lys(256)/Lys(257) double mutant. Binding of bovine Pg (BPg) to the SK.Pm complex containing fluorescently labeled Pm demonstrated LBS-dependent assembly of a SK.labeled Pm.BPg ternary complex, whereas BPg did not bind to the complex containing the SK Lys(256)/Lys(257) mutant. BPg was activated by SK.Pm with a K(m) indistinguishable from the K(D) for BPg binding to form the ternary complex, whereas the SK Lys(256)/Lys(257) mutant did not support BPg activation. We conclude that SK residues Arg(253), Lys(256), and Lys(257) mediate Pg substrate recognition through kringle 5 of the [Lys]Pg and mini-Pg substrates. A molecular model of the SK.kringle 5 complex identifies the putative interactions involved in LBS-dependent Pg substrate recognition.