The esterolytic activity of bovine .alpha.-thrombin on the synthetic substrate N-.alpha.-p-tosyl-L-arginine methyl ester (TosArgOMe) was stimulated when the prothrombin activation fragment, prothrombin fragment 2, was added as previously reported. A similar stimulation of .beta.-thrombin was observed upon addition of prothrombin fragment 2. The dissociation constant was 7.7 .times. 10-10 M and there was 1 molecule of prothrombin fragment 2 bound per molecule of .alpha.-thrombin. Prethrombin-2 competed for prothrombin fragment 2, so the enhancement of the esterolytic activity of .alpha.-thrombin by prothrombin fragment 2 was used as a probe to determine the dissociation constant for the binding of prothrombin fragment 2 to prethrombin 2. The dissociation constant for this association was 1.3 .times. 10-10 M. The kinetic parameters for the reaction of .alpha.-thrombin on TosArgOMe were determined in the absence and presence of prothrombin fragment 2. In the absence of prothrombin fragment 2, Km(app) = 1.92 .times. 10-4 M, and k3(app) = 35.8 mol of TosArgOMe/mol of .alpha.-thrombin s-1; in the presence of prothrombin fragment 2, Km(app) = 1.76 .times. 10-4 M, and k3(app) = 60.5 mol of TosArgOMe/mol of .alpha.-thrombin s-1. The stimulatory effect of bovine prothrombin fragment 2 on bovine .alpha.-thrombin was reflected in k3(app) and not in Km(app). In contrast to the stimulatory effect of prothrombin fragment 2 on the thrombin-catalyzed hydrolysis of TosArgOMe, it inhibited the activity of .alpha.-thrombin toward N-.alpha.-benzoyl-L-arginine ethyl ester and N-.alpha.-benzoyl-L-arginine p-nitroanilide. The inhibition of activity toward these substrates by prothrombin fragment 2 was also reflected in k3(app). Activity toward the nonspecific substrate p-nitrophenyl butyrate was completely inhibited by the addition of prothrombin fragment 2. Prothrombin fragment 2 had no effect on the inhibition of .alpha.-thrombin activity by the active-site serine inhibitors diisopropyl phosphofluoridate, phenylmethanesulfonyl fluoride, or p-nitrophenyl guanidinobenzoate. Inhibition by the active-site-histidine-modifying inhibitor, N-.alpha.-p-tosyl-L-arginine chloromethyl ketone, was enhanced by the addition of prothrombin fragment. Soybean trypsin inhibitor reduced the stimulation by prothrombin fragment 2, but only at high molar ratios. Prothrombin fragment 2 had no effect on the clotting activity of .alpha.-thrombin, nor inhibition of this activity by heparin, hirudin, or diisopropyl phosphofluoridate. Bovine prothrombin fragment 2 enhanced the esterolytic activity of both human and bovine .alpha.-thrombin, but human prothrombin fragment 2 did not enhance the esterolytic activity of either human or bovine .alpha.-thrombin.