Aging alters cellular responses to both heat and oxidative stress. Thiol-mediated metabolism of reactive oxygen species (ROS) is believed to be important in aging. To begin to determine the role of thiols in aging and heat stress, liver glutathione (GSH) was depleted by L-buthionine sulfoximine (BSO) administration in young (6 mo) and old (24 mo) Fisher 344 rats prior to heat stress. Animals were given BSO (4 mmol/kg i.p.) or saline (1 ml i.p.) 2 hr before heat stress, and subsequently heated to a core temperature of 41°C over a 90-min period. Liver tissue was collected prior to, and 0, 30, and 60 min after heat stress. BSO inhibited glutamate cysteine ligase (GCL: the rate limiting enzyme in GSH synthesis) catalytic activity and resulted in a decline in liver GSH and GSSG that was more pronounced in young compared to old animals. Catalase activity did not change between groups until 60 min after heat stress in young BSO-treated rats. Young animals experienced a substantial and persistent reduction in CuZnSOD activity with BSO treatment. MnSOD activity increased with BSO but declined after heat stress. The differences in thiol depletion observed between young and old animals with BSO treatment may be indicative of age-related differences in GSH compartmentalization that could impact upon maintenance of redox homeostasis and antioxidant balance immediately after a physiologically relevant stress. The significant changes in antioxidant enzyme activity after GSH depletion suggest that thiol status can influence the regulation of other antioxidant enzymes.