The inactivation of thymidylate synthase (5,10-methylene-tetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) by a number of disulfides has been examined and found to be a second-order process. The apparent second-order rate constant was strongly influenced by the chemical structure of the disulfide. The data suggest that negatively charged functional groups decrease the reactivity of the disulfides and that positively charged groups enhance the reactivity. GSSG did not react with non-catalytic SH groups, since the number of SH groups of both GSSG-treated and untreated thymidylate synthase was the same. Several sulfhydryl compounds were tested for their ability to reactivate thymidylate synthase that had been inhibited by 2,2'-dithiodipyridine. Complete reactivation was obtained with either dithiothreitol or 2-mercaptoethanol. Reactivation by 2-mercaptoethanol was a second-order process.