A disulfide bond between key redox-sensitive cysteine residues and glutathione is one mechanism by which redox related allosteric effectors can regulate protein structure and function. Here we test the hypothesis that glutaredoxin-1 (Grx-1), a member of the oxidoreductase family of enzymes, may be a critical component of redox-sensitive molecular switches by mediating reversible protein S-glutathionylation and enzymatic catalysis of thiol/disulfide exchange. Deglutathionylation of a 70 kDa protein by Grx-1 was detected using a monoclonal antibody specific to protein S-glutathionylation. Heat shock cognate protein 70 (Hsc70) was identified as a substrate of Grx-1 through mass spectrometry. Recombinant Hsc70 was glutathionylated in vitro, and protein S-glutathionylation reversed by Grx-1. Glutathionylated Hsc70 was more effective in preventing luciferase aggregation at 43°C than reduced Hsc70 in a dose dependent fashion. ATP did not effect the chaperone activity of Hsc70-SG but did increase the activity of reduced Hsc70-SG. Reversible glutathionylation of Hsc70 may provide a mechanism for post-translation regulation of chaperone activity.