Reaction of rabbit muscle pyruvate kinase with the affinity label 5'-[p-(fluorosulfonyl) benzoyl] guanosine (5'-FSBG), at pH 7.65 and 7.93, leads to a loss in enzyme activity. The inactivation is characterized by a biphasic kinetic profile, with the initial phase accounting for approximately 55% of the reduction in enzymatic activity. For both the rapid and slow phases, at pH 7.93, the inactivation rate constants are linearly proportional to the reagent concentration (from 0.48 to 3.0 mM), yielding second-order rate constants of 195 min-1 M-1 and 19 min-1 m-1, respectively. The effect of ligands was tested on the two phases of inactivation. For both, a decrease in the inactivation rate was produced by Mg2+ alone, but the best protection was provided by Mg2+ plus either ADP or GDP, suggesting that the reaction occurs in the region of the metal-nucleotide binding site. Modified pyruvate kinase is completely reactivated by incubation with 20 mM dithiothreitol, indicating the involvement of cysteine in the inactivation, indicating the involvement of cysteine in the inactivation process. Reaction with [5'=3H]-5'-FSBG leads to the incorporation of up to 1.3 mol of radioactive reagent per mol of enzyme subunit; however, identical radiolabel incorporation is observed before or after dithiothreitol reactivation of modified enzyme. This result implies that the labeled amino acid residue, measured by means of incorporation, is not directly involved in the inactivation process. In contrast, inactivation was found to correlate well with the loss of two free sulfhydryl groups per enzyme subunit and the restoration of activity to correlate with the regeneration of two free sulfhydryls after treatment of modified enzyme with dithiothreitol. It is proposed that inactivation of pyruvate kinase by 5'-FSBG proceeds by formation of thiol sulfonate followed by a rapid displacement of the sulfinic acid moiety by a second cysteine to yield a disulfide. A negative cooperatively in the interaction of pyruvate kinase subunits with 5'-[p-(fluorosulfonyl)-benzoyl] guanosine might best account for the biphasic inactivation kinetics.