Cells can respond to a sublethal oxidative stress by up-regulating their intracellular glutathione (GSH) pool. Such increased GSH concentration is likely to be protective against further oxidative challenge, and, in fact, pre-exposure to low levels of oxidants confers increased cellular resistance to subsequent greater oxidative stress. Previously, we have shown that pretreatment of rat lung epithelial L2 cells with sublethal concentrations of tert-butylhydroquinone (TBHQ) increases intracellular GSH concentration in a concentration- and time-dependent manner. This increase resulted from up- regulation of both γ-glutamyltranspeptidase (GGT) and γ-glutamylcysteine synthetase (GCS). Therefore, we investigated whether such increased GSH concentration protected these cells against a subtle loss in function caused by a subsequent challenge with sublethal concentrations of tert-butyl hydroperoxide (tBOOH) (≤200 μM), mimicking a physiological oxidative stress. Activation of L2 cell purinoreceptors with 100 μM AD caused an elevation of intracellular Ca2+. This response was suppressed by a brief pre-exposure to tBOOH. The inhibition, however, was alleviated dramatically by a 16-hr pretreatment with 50 μM TBHQ. The same TBHQ pretreatment also protected the cells from ATP-depletion induced by tBOOH. L-Buthionine S,R- sulfoximine (BSO), an irreversible inhibitor of GCS, prevented the increase in intracellular GSH and also completely removed the protection by TBHQ in maintaining the ATP level. Thus, pre-exposure to a sublethal level of TBHQ results in protection of cell functions from hydroperoxide toxicity. This protection appears to depend on alteration of the intracellular GSH pool, the modulation of which constitutes an adaptive response to oxidative stress.