Nitrophorin-2 (NP-2), isolated from salivary glands of the blood-sucking insect Rhodnius prolixus, has been shown to be a specific inhibitor of the intrinsic factor X-(FX)-activating complex. The inhibitory effect of NP-2 is most potent in the presence of both FVIIIa and phospholipids (artificial phospholipid vesicles or activated human platelets). Detailed kinetic analyses of the inhibitory mechanism of NP-2 demonstrated a decrease in both Vmax and K(m) of activated FIX-(FIXa)-catalyzed FX activation in the presence of FVIIIa and phospholipid vesicles, characteristic of a hyperbolic mixed-type reversible inhibitor. NP-2 exhibits a higher binding affinity for the enzyme-substrate complex, i.e., FIXa/FVIIIa/ Ca2+/phospholipids/FX complex (Ki' = 6.2 nM) than for the enzyme complex, i.e., FIXa/FVIIIa/Ca2+/ phospholipids (Ki = 16.5 nM). The same inhibitory kinetic mechanism is valid in platelet-mediated FIXa-catalyzed FX activation (Ki' = 5.9 nM and Ki = 12.6 nM, respectively). The fact that NP-2 increases the concentrations (EC50) of FIXa, FVIIIa, and phospholipid vesicles required for half-maximal rates of FX activation suggests that NP-2 interferes with the functioning of all three major components of the intrinsic FX-activating complex. NP-2 was found to inhibit FX activation when either phospholipids or FVIIIa are present, but not in the absence of both factors. Taken together, we conclude that NP-2 is a unique, potent, and highly specific inhibitor of the intrinsic FX-activating complex that inhibits FIXa bound either to the phospholipid or activated platelet surface or to the cofactor FVIIIa by interfering with the assembly of FX-activating complex on these surfaces.