Activation of Glu- and Lys-plasminogen by various concentrations of recombinant staphylokinase (SAK) were studied by the generation of amidolytic activity from the chromogenic substrate S-2251(H-D-Val-Leu-Lys-pNA) and by SDS-PAGE analysis. Surprisingly, excess SAK decreased and fixed the rate of S-2251 hydrolysis in a mixture of Lys-plasminogen and SAK. Since the effect of SAK on S-2251 hydrolysis by plasma was similar, the hydrolysis kinetics by free plasmin and plasmin-SAK complex were studied. Hydrolysis by either enzyme form followed Michaelis-Menten kinetics with a Km of 0.38 mM for plasma and 3.74 mM for SAK-plasmin complex. The catalytic rate constant was 22.7 s-1 for plasmin and 21.0 s-1 for the SAK-plasmin complex. With excess SAK and vigorous removal of plasmin activity from plasminogen, the pre-activation lag period differed greatly between Glu- and Lys-plasminogen. Based on the different substrate specificity of plasmin and plasmin-SAK complex, we analyzed the Glu-plasminogen activation with either catalytic or excess SAK. With excess SAK, almost no Lys-plasminogen was detectable and whole Glu-plasminogen was converted directly to Glu-plasmin, then gradually to Lys-plasmin. In contrast, Lys-plaminogen appeared rapidly with catalytic amount of SAK. These results suggest that inhibition of Glu-plasminogen to Lys-plasminogen to Lys-plasminogen conversion in the plasminogen-SAK complex in the presence of excess SAK prolonged the initial lag phase of activation.