Deuterium kinetic solvent isotope effects for the human alpha-thrombin-catalyzed hydrolysis of (1) substrates with selected P-1-P-3 sites, Z-Pro-Arg-7-amido-4-methylcoumarin (7-AMC), N-t-Boc-Val-Pro-Arg-7-AMC, Bz-Phe-Val-Arg-4-nitroanilide (pNA), and H-D-Phe-L-Pip-Arg-pNA, are (DOD)k(cat) = (2.8-3.3) +/- 0.1 and (DOD)(k(cat)/K-m) = (0.8-2.1) +/- 0.1 and (2) internally fluorescence-quenched substrates (a) (AB)Val-Phe-Pro-Arg-Ser-Phe-Arg-Leu-Lys(DNP)-Asp-OH, an optimal sequence, and (b) (AB)Val-Ser-Pro-Arg-Ser-Phe-Gln-Lys(DNP)-Asp-OH, recognition sequence for factor VIII, are (DOD)k(cat) = 2.2 +/- 0.2 and (DOD)(k(cat)/K-m) = (0.8-0.9) +/- 0.1, at the pL (L = H, D) maximum, 8.4-9.0, and (25.0-26.0) +/- 0.1 degreesC. The most plausible models fitting the partial isotope effect (proton inventory) data have been selected on the basis of lowest values of the reduced chi squared and consistency of fractionation factors at all substrate concentrations, assuming rate-determining acylation. The data for Z-Pro-Arg-7-AMC are consistent with a single-proton bridge at the transition state phi(TS) = 0.39 +/- 0.05 and components for solvent reorganization phi(S) = 0.8 +/- 0.1 and phi(S) = 1.22 for k(cat) and k(cat)/K-m, respectively. The data for tripeptide amides fit bowl-shaped curves; an example is N-t-Boc-Val-Pro-Arg-7-AMC: phi(1)(TS) = phi(2)(TS) = 0.57 +/- 0.01 and phi(S) = 1 for k(cat) and 1.6 +/- 0.1 for k(cat)/K-m. Proton inventories for the nonapeptide (2b) are linear. The data for k(cat) for H-D-Phe-L-Pip-Arg-pNA and the decapeptide (2a) are most consistent with two identical fractionation factors for catalytic proton bridging, phi(1)(TS) = phi(2)(TS) = 0.68 +/- 0.02 and a large inverse component (phi(S) = 3.1 +/- 0.5) for the latter, indicative of substantial solvent reorganization upon leaving group departure. Proton inventory curves for k(cat)/K-m for nearly all substrates are dome-shaped with an inverse isotope effect component (phi(S) = 1.2-2.4) originating from solvent reorganization during association of thrombin with substrate. These large contributions from medium effects are in full accord with the conformational adjustments required for the fulfillment of the dual, hemostatic and thrombolytic, functions of thrombin.