Abstract S-Glutathionylation (thiolation) is a ubiquitous redox-sensitive and reversible modification of protein cysteinyl residues that can directly regulate their activity. While well established in animals, little is known about the formation and function of these mixed disulfides in plants. After labeling the intracellular glutathione pool with [35S]cysteine, suspension cultures of Arabidopsis (Arabidopsis thaliana ecotype Columbia) were shown to undergo a large increase in protein thiolation following treatment with the oxidant tert-butylhydroperoxide. To identify proteins undergoing thiolation, a combination of in vivo and in vitro labeling methods utilizing biotinylated, oxidized glutathione (GSSG-biotin) was developed to isolate Arabidopsis proteins/protein complexes that can be reversibly glutathionylated. Following two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry proteomics, a total of 79 polypeptides were identified, representing a mixture of proteins that underwent direct thiolation as well as proteins complexed with thiolated polypeptides. The mechanism of thiolation of five proteins, dehydroascorbate reductase (AtDHAR1), zeta-class glutathione transferase (AtGSTZ1), nitrilase (AtNit1), alcohol dehydrogenase (AtADH1), and methionine synthase (AtMetS), was studied using the respective purified recombinant proteins. AtDHAR1, AtGSTZ1, and to a lesser degree AtNit1 underwent spontaneous thiolation with GSSG-biotin through modification of active-site cysteines. The thiolation of AtADH1 and AtMetS required the presence of unidentified Arabidopsis proteins, with this activity being inhibited by S-modifying agents. The potential role of thiolation in regulating metabolism in Arabidopsis is discussed and compared with other known redox regulatory systems operating in plants.