Low molecular weight thiol/disulfide redox pools are dependent upon extracellular cysteine (Cys) availability. We determined whether dietary sulfur amino acid (SAA) deficiency induces oxidative stress in vivo, as determined by redox state of major thiol/disulfide couples in plasma [Cys/cystine (CySS)] and intestinal mucosa [glutathione (GSH)/glutathione disulfide (GSSG)]. Rats were fed isocaloric, isonitrogenous semipurified diets: either SAA-adequate (control), SAA-deficient, or SAA-supplemented, pair-fed to intake of the SAA-deficient group. Reference rats consumed standard rat food ad libitum. After 7 d, plasma and gut mucosal samples were analyzed for Cys, CySS, GSH and GSSG, and the redox potentials of Cys/CySS and GSH/GSSG were determined. Mean daily food intake in the pair-fed rats was similar (approximately one-half of reference-rat intake). Body weight decreased in all pair-fed groups, but rats fed the SAA-deficient diet lost significantly more body weight. Dietary SAA deficiency decreased GSH concentrations in both plasma and gut mucosa, increased plasma GSSG, and oxidized plasma and gut mucosal GSH/GSSG redox and plasma Cys/CySS redox. SAA supplementation resulted in a more reducing plasma Cys/CySS redox potential. Reference rats exhibited similar tissue and plasma GSH/GSSG redox as rats that ate semipurified SAA-adequate rat food, which provided similar net SAA intake. Our in vivo data show that inadequate dietary SAA intake oxidizes the thiol/disulfide redox status in rat-gut mucosa and plasma. Such oxidation of redox pools is associated with oxidative stress and the onset or progression of several pathological conditions. Thus, dietary SAA deficiency could contribute to the progression of disease by causing an oxidation of these components.