The feasibility of a new approach to incorporation of spectroscopic probes into the active sites of certain serine proteases has been demonstrated. The method is based on inactivation of a serine protease with a thioester derivative of a peptide chloromethyl ketone. The thiol group generated by reaction of the covalent enzyme-inhibitor complex with NH2OH provides a unique site for subsequent labeling with thiol-reactive probes. To evaluate the method, N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl was synthesized by reaction of the thrombin-specific tripeptide chloromethyl ketone with succinimidyl (acetylthio)acetate and purified by sulfopropyl-Sephadex and Sephadex G-10 chromatography. Reverse-phase high-performance liquid chromatography indicated that the product was 90 +/- 3% pure. The compound was quantitated by using 5,5'-dithiobis(2-nitrobenzoic acid) to measure the concentration of thiol produced in the presence of NH2OH. On this basis, titrations of the irreversible loss of human alpha-thrombin activity had end points of 1.1 +/- 0.1 mol of inhibitor/mol of active sites, indicating a 1:1 stoichiometry for inactivation. Incubation of N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-thrombin with 5-(iodoacetamido)fluorescein in the presence of NH2OH resulted in incorporation of 0.96 mol of the fluorescence probe/mol of active sites and the appearance of fluorescein fluorescence associated with the active site containing B-chain on sodium dodecyl sulfate-polyacrylamide gels. Fluorescence labeling of thrombin required reaction of the inhibitor at the active site as well as subsequent generation of the thiol group with NH2OH. It is concluded that active site selective labeling can be achieved by using this approach, which is likely to be applicable to other proteases, peptide chloromethyl ketones, and a wide variety of probes.