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Human thrombin is inactivated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) by a second order process. A plot of the pseudo-first order rate constant for inactivation by 20 mM EDC at different pH values from 4.2 to 7.7 at 25 degrees C shows that this inactivation is due to the modification of a protonated carboxyl with a pKa of 5.6. The rate of inactivation by EDC at pH 6.0 is reduced, but not eliminated by saturating levels of the competitive inhibitor dansyl-L-arginine N-(3-ethyl-1,5-pentanediyl)amide, suggesting that the essential carboxyl modified is one other than Asp-189 in the substrate specificity pocket of thrombin. Complete inactivation by 14C-EAC correlates with the incorporation of approximately 2.5-3 EACs per thrombin.