The intrinsic factor Xase complex (FXase) is comprised of a serine protease, FIXa, and a protein cofactor, FVIIIa, assembled on a phospholipid surface. Activity of FXase decays with time and reflects the lability of FVIIIa. Two mechanisms potentially contribute to this decay: (i) a weak affinity interaction between the FVIIIa A2 subunit and Al/A3-Cl-C2 dimer and (ii) FVIIIa inactivation resulting from FIXa-catalyzed proteolysis of the Al subunit. At low reactant concentrations (0.5 nm FVIIIa; 5 nm FIXa), FXase decay is governed by the inter-FVIIIa subunit affinity and residual activity approaches a value consistent with this equilibrium, as judged by reactions containing exogenous A2 subunit. Analysis using a mutant form of FVIII (FVIIIR336I) possessing an altered FIXa cleavage site, showed similar rates of FXase decay (0.12 min(-1)) and confirmed the lack of contribution of proteolysis under these conditions. When the concentration of FIXa was increased 10-fold, the initial rate of decay of FXase containing native FVIIIa increased (0.82 min(-1)) and paralleled the rate of proteolysis of Al subunit. However, the rate of decay of FXase containing the FVIIIaR336I was reduced (0.048 min(-1)) consistent with the elevated concentration of FIXa stabilizing the labile subunit structure of the cofactor. Reconstitution of FVIII with FIXa-cleaved light chain showed that cleavage at the alternate FIXa site (A3 domain) was not inhibitory to FXase. The presence of substrate FX resulted in a 10-fold reduction in the rate of FIXa-catalyzed proteolysis of FVIIIa. These results suggest a model whereby decay of FXase results from both FVIIIa subunit dissociation and FIXa-catalyzed cleavage, dependent upon the relative concentration of reactants, with greater contribution of the former at low values and, in the absence of substrate, greater contribution of the latter at high values.