The kinetic mechanism of the inhibition of alpha-thrombin by hirudin was analyzed using the hirudin-derived fragments hirudin(1-47) and hirudin(45-65). Previously, these fragments have been shown to interact with alpha-thrombin at distinct sites inhibiting thrombin-mediated clot formation. Binding to the active site the N-terminal fragment hirudin(1-47) competitively inhibits hydrolysis of the substrates Tos-Gly-Pro-Arg-NH-Mec (Tos, tosyl; NH-Mec, 4-methylcoumaryl-7-amide) and fibrinogen with Ki values of 420 +/- 18 nM and 460 +/- 25 nM, respectively. Interacting with the anion-binding site of alpha-thrombin the C-terminal fragment competitively inhibits the hydrolysis of fibrinogen with a Ki of 760 +/- 40 nM. It was found, however, that this fragment acts as a hyperbolic uncompetitive inhibitor with respect to the hydrolysis of the peptide-NH-Mec substrate. According to the Botts-Morales scheme for enzyme inhibition, the parameters Ki = 710 +/- 38 nM, K'i = 348 +/- 22 nM, as well as alpha = beta = 0.49 of thrombin inhibition by the C-terminal fragment hirudin(45-65), were obtained. The results are discussed in terms of the interaction of hirudin and thrombin.