Studies were performed in rats that had been fasted 24 h, fed a glutathione (GSH)-free semisynthetic diet (AIN-76), and fed the same diet supplemented with GSH. The results from the fasted rats and those fed GSH-free diet showed that the duodenum and jejunum contained 0.2-0.5 mumol of GSH/gram wet wt of luminal contents. The GSH contents of biliary juice was sufficient to maintain this amount of GSH in the intestinal lumen. Other analyses showed that cell sloughing, bacterial GSH content, and GSH secretion by epithelial cells of the jejunum were not sufficient to account for this content. GSH concentrations following consumption of a GSH-supplemented diet (5-50 mg/g AIN-76) showed a rapid increase in all regions of the small intestine and indicated that removal occurred primarily in the jejunum. However, the combined activities of brush-border gamma-glutamyltransferase and GSH uptake systems were not sufficient to remove all of the ingested GSH. Results from in situ vascular perfusions of small intestine showed that the upper jejunum is a principal site of GSH absorption. Measurements of the GSH-to-glutathione disulfide (GSSG) ratio in the lumen after ingestion of GSSG (5 mg/g diet) indicated that the upper small intestine also has a mechanism for reducing GSSG to GSH. The results therefore indicate that GSH is present in the lumen of the small intestine of rat under most if not all conditions. Although the physiological importance of luminal GSH remains unclear, it could potentially be used to detoxify reactive electrophiles in the diet or be absorbed for intracellular detoxication reactions.