The reduction of the platinum(IV) prodrug trans,trans,trans-[PtCl2(OH)(2)(c-C6H11NH2)(NH3)] (JM335) by L-cysteine, DL-penicillamine, DL-homocysteine, N-acetyl-L-cysteine, 2-mercaptopropanoic acid, 2-mercaptosuccinic acid, and glutathione has been investigated at 25 degrees C in a 1.0 M aqueous perchlorate medium with 6.8 less than or equal to pH less than or equal to 11.2 using stopped-flow spectrophotometry. The stoichiometry of Pt(IV):thiol is 1:2, and the redox reactions follow the second-order rate law -d[Pt(IV)]/dt = k[Pt(IV)][RSW](tot), where k denotes the pH-dependent second-order rate constant and [RSH](tot) the total concentration of thiol. The pH dependence of k is ascribed to parallel reductions of JM335 by the various protolytic species of the thiols, the relative contributions of which change with pH. Electron transfer from thiol (RSH) or thiolate (RS-) to JM1335 is suggested to take place as a reductive elimination process through an attack by sulfur at one of the mutually trans chloride ligands, yielding trans-[Pt(ON)(2)-(c-C6H11NH2)(NH3)] and RSSR as the reaction products, as confirmed by H-1 NMR. Second-order rate constants for the reduction of JM335 by the various protolytic species of the thiols span more than 3 orders of magnitude. Reduction with RS- is similar to 30-2000 times faster than with RSH. The linear correlation log(k(RS-)) = (0.52 +/- 0.06)-pK(RSH) - (2.8 +/- 0.5) is observed, where k(RS-) denotes the second-order rate constant for reduction of JM335 by a particular thiolate RS- and K-RSH is the acid dissociation constant for the corresponding thiol RSH. The slope of the linear correlation indicates that the reactivity of the various thiolate species is governed by their proton basicity, and no significant steric effects are observed. The half-life for reduction of JM335 by 6 mM glutathione (40-fold excess) at physiologically relevant conditions of 37 degrees C and pH 7.30 is 23 s. This implies that JM335, in clinical use, is likely to undergo in vivo reduction by intracellular reducing agents such as glutathione prior to binding to DNA. Reduction results in the immediate formation of a highly reactive platinum(II) species, i.e., the bishydroxo complex in rapid protolytic equilibrium with its aqua form.