Activated porcine Factor IX is irreversibly inhibited by an active site histidine-directed serine protease inhibitor, dansyl-glutamyl-glycyl-arginyl-chloromethylketone (DEGR-CK). The kinetics of inhibition are second order up to inhibitor concentrations of 10(-5) M. The apparent second-order rate constant (in 0.20 M NaCl, pH 8.0) is 1.7 X 10(4) M-1 min-1, which is considerably lower than values reported for Factor Xa, thrombin, plasmin, and kallikrein. Reaction of increasing concentrations of DEGR-CK with Factor IXa, followed by analysis of residual enzymatic activity, yields 1.2 mol DEGR-CK/mol protein, indicating 1:1 stoichiometry for the DEGR-CK/Factor IXa interaction. DEGR-Factor IXa is a potent anticoagulant in vitro. A concentration of 1 nM causes 50% inhibition of the ability of normal porcine-citrated plasma to correct either Factor VIII- or Factor IX-deficient plasmas (intrinsic pathway factors). In contrast, more than 100 nM DEGR-Factor IXa is required to cause 50% inhibition of Factor VII (extrinsic pathway) or Factor X (common pathway) assays. Activation of porcine Factor VIII:C by thrombin in the presence of DEGR-Factor IXa and phosphatidylcholine-phosphatidylserine vesicles reveals that DEGR-Factor IXa markedly stabilizes the spontaneous loss of Factor VIII:Ca activity as does unmodified Factor IXa [P. Lollar, G.J. Knutson, and D. N. Fass (1984) Blood 63, 1303-1308]. These results suggest that DEGR-Factor IXa incorporates into the intrinsic pathway Factor X-activator enzymatic complex, and also that stabilization of Factor VIII:Ca by this complex is independent of the active site of Factor IXa. Inhibition of Factor IXa by DEGR-CK results in the first reported irreversible active-site-modified derivative of this enzyme. DEGR-CK promises to be a useful reagent in the study of the Factor X activator complex. Conceivably, its specific anticoagulant properties could have future clinical benefit.