The activation of human plasminogen by a highly purified staphylokinase was investigated using casein or an active site titrant (p-nitrophenyl-p-guanidinobenzoate, NPGB) as a substrate. The reaction rate was time dependent, showing a pronounced lag period with either substrate. Saturation curve estimated from the caseinolytic assay was sigmoid, but changed to quasi-hyperbolic in the presence of pre-formed human plasmin. With NPGB, the extent of plasminogen conversion into esterolytic plasmin was directly proportional to staphylokinase concentration, and the saturation point was reached when the molar concentration of staphylokinase equaled that of plasminogen. It is concluded that staphylokinase acts stoichiometrically, forms an equimolar complex with plasminogen, and thus is not an enzyme but a modifier. Staphylokinase-activated plasminogen exhibits properties of a hysteretic enzyme.