L-Galactonolactone oxidase was found to be inactivated by various sulfhydryl reagents: the order of inactivation rate was HgCl2 greater than p-chloromercuribenzoate greater than 4,4'-dipyridyldisulfide greater than 2,2'-dipyridyldisulfide greater than 5,5'-dithio-bis-(2-nitrobenzoate) greater than N-ethylmaleimide greater than iodoacetamide. The inactivation by 4,4'dipyridyldisulfide was studied in detail, and it was found that the maximum degree of inactivation attained with increasing reagent concentration was 93%, and that the kinetics of inactivation was first order with respect to the reagent concentration. The pH dependence of the second-order rate constant of the inactivation revealed that sulfhydryl group with a pKa of approximately 9.8 was involved in the inactivation process. The value of pKa is high compared with that of low-molecular weight thiols, indicating that the ionization of the sulfhydryl group is affected by the electric field of a negatively charged group located in its vicinity. The values of Km and Vmax for the L-galactonolactone oxidase reaction did not change greatly around pH 9.8. This finding is consistent with the view that the sulfhydryl group does not participate in the catalytic process. The velocity of inactivation in the presence of substrate was considerably greater than that observed in its absence. It appears that the sulfhydryl group becomes more reactive during the catalytic cycle of the enzyme. It was also noted that the absorption spectrum of the flavin prosthetic group (the oxidized form) was modified by adding p-chloromercuribenzoate.