Redox control of the transcription factor c‐Jun maps to a single cysteine in its DNA binding domain. However, the nature of the oxidized state of this cysteine and, thus, the potential molecular mechanisms accounting for the redox regulation of c‐Jun DNA binding remain unclear. To address this issue, we have analyzed the purified recombinant c‐Jun DNA binding domain for redox‐dependent thiol modifications and concomitant changes in DNA binding activity. We show that changes in the ratio of reduced to oxidized glutathione provide the potential to oxidize c‐Jun sulfhydryls by mechanisms that include both protein disulfide formation and S‐glutathiolation. We provide evidence that S‐glutathiolation, which is specifically targeted to the cysteine residue located in the DNA binding site of the protein, may account for the reversible redox regulation of c‐Jun DNA binding. Furthermore, based on a molecular model of the S‐glutathiolated protein, we discuss the structural elements facilitating S‐glutathiolation and how this modification interferes with DNA binding. Given the structural similarities between the positively charged cysteine‐containing DNA binding motif of c‐Jun and the DNA binding site of related oxidant‐sensitive transcriptional activators, the unprecedented phenomenon of redox‐triggered S‐thiolation of a transcription factor described in this report suggests a novel role for protein thiolation in the redox control of transcription.—Klatt, P., Molina, E. P., de Lacoba, M. G., Padilla, C. A., Martínez‐Galisteo, E., Bárcena, J. A., Lamas, S. Redox regulation of c‐Jun DNA binding by reversible S‐glutathiolation. FASEB J. 13, 1481–1490 (1999)