Here, we report on the facilitated reactivation (85%) of oxidatively inactivated rhodanese by an oxidized form of the molecular chaperone GroEL (ox-GroEL). Reactivation by ox-GroEL required a reductant, and the enzyme substrate, sodium thiosulfate. Also, we found that ox-GroEL formed a complex with oxidatively inactivated rhodanese as shown by differential centrifugation and fluorescence spectroscopy. Ox-GroEL was obtained upon incubation of native GroEL for 16 h with 5 mM hydrogen peroxide. Under these conditions, GroEL was shown to retain its quaternary and secondary structures, but it displayed an increased exposure of hydrophobic surfaces as detected with 1,1'-bis(4-anilino) naphthalene-5,5'-disulfonic acid (bisANS) fluorescence. Additionally, ox-GroEL was significantly more sensitive towards proteolysis with trypsin compared to the native form of the protein. The oxidatively inactivated form of rhodanese, also had an increased exposure of hydrophobic surfaces, as previously reported. Thus, the proteins binding appeared to be mediated by hydrophobic interactions. Unlike in prior reactivation studies that involved native GroEL or alpha-crystallin, we have clearly shown that an oxidized form of GroEL can function as a molecular chaperone in the reactivation of oxidatively inactivated rhodanese suggesting that GroEL retains the ability to protect proteins during oxidative stress.