Although there has been considerable interest in the regulation of NF kappa B activation by glutathionylation, the possibility Of I kappa B as a target for glutathionylation has not been investigated. We now report that Cys(189) Of I kappa B alpha is a target for S-glutathionylation. This modification is reversed by thiols such as dithiothreitol and GSH. The glutathionylated I kappa B alpha. appears to be significantly less susceptible than is native protein to phosphorylation by I kappa B kinase and casein kinase II, as well as to in vitro ubiquitination. This finding suggests that glutathionylation plays a regulatory role, presumably through structural alterations. HeLa cells treated with oxidant inducing GSH oxidation such as diamide showed the accumulation of glutathionylated I kappa B alpha. This mechanism suggests an alternative modification to the redox regulation of cysteine in I kappa B alpha and a possible mechanism in the regulation of NF kappa B activation. (c) 2008 Elsevier Inc. All rights reserved.