The enzymatic activity of hexokinase (ATP : D-hexose 6-phosphotransferase, EC 2.7.1.1) decreased rapidly when the enzyme was exposed to the lactoperoxidase antimicrobial system (consisting of lactoperoxidase, H2O2 and SCN-). Inactivation did not begin until the reaction of one sulfhydryl group per hexokinase monomer was completed. Loss of enzyme activity accompanied the reaction of at least one additional sulfhydryl group per monomer. Covalent incorporation of 14C-labeled SCN- into hexokinase increased as the inactivation reaction progressed. The rate of the hexokinase activity loss dependent on temperature, pH and the presence of glucose and phosphate ion. When H2O2 and SCN- were applied to a Sepharose column bearing covalently attached lactoperoxidase, the column eluate inactivated hexokinase. This demonstrated that the lactoperoxidase molecule itself need not be in contact with hexokinase in order to catalyze hexokinase inactivation. The sulfhydryl-reactive oxidation product of SCN- which is generated by the column is sufficient. The results are consistent with a two-stage reaction in which the exposed, non-essential sulfhydryl groups on the hexokinase molecule react first to produce an enzymatically active but unstable form of hexokinase. This modified form of hexokinase then undergoes a spontaneous, temperature-dependent structural change, which allows reaction of previously shielded, essential sulfhydryl groups. The phenomenon described here suggests a possible mechanism for the antimicrobial effects of the lactoperoxidase system.