The kinetics of activation of both [Glu1]- and [Lys78]Plg(S741C-fluorescein by native (recombinant) tissue-type plasminogen activator and its deletion variants lacking either the finger or kringle-2 domain were measured by fluorescence within fully polymerized fibrin clots. The kinetics conform to the Michaelis-Menten equation at any fixed fibrin concentration so long as the plasminogen concentration is expressed as either the free or fibrin-bound, but not the total. The apparent kcat and Km values both vary systematically with the concentration of fibrin. Competition kinetics disclosed an active site-dependent interaction between t-Pa and [Glu1]Plg(S741C-fluorescein) in the presence, but not the absence, of fibrin. A steady-state template model having the rate equation v/[A]o = kcat(app).[Plg]/(Km(app) + [Plg]) was derived and used to interpret the data. The model indicates that catalytic efficiency is determined by the stability of the ternary activator-fibrin-plasminogen complex rather than the binding of the activator or plasminogen to fibrin. This implies that efforts to improve the enzymatic properties of t-PA might be more fruitfully directed at enhancing the stability of the ternary complex rather than fibrin binding.