Cellular damage from reactive intermediates formed during xenobiotic biotransformation is prevented by the presence of adequate levels of antioxidant chemicals in the tissues. Equally important for cell protection is the rate at which these chemicals are replaced if tissue stores are depleted. The present experiments, using adult male Sprague-Dawley rats and Hartley guinea pigs, were conducted to ascertain what effects mainstream (MS) and sidestream (SS) tobacco smoke would have on the water-soluble, cytoplasmic antioxidants, ascorbic acid (AA) and reduced glutathione (GSH). The animals were exposed by nose-only inhalation to varying doses (40, 120, 240 puffs) of a 1:5 dilution of a 35-ml volume of freshly generated MS from cigarettes made from different types of tobacco and delivered by a B.-A.T-Mason inhalation apparatus. The animals were euthanized either immediately following exposure or at 3 and 6 h. The blood, lungs, liver, kidneys, heart and bladder were removed for the quantitation of AA and GSH following homogenization and deproteinization. Immediately following exposure to MS, dose-dependent decreases in pulmonary and renal GSH were observed in rats whereas, in guinea pigs, reductions in pulmonary, hepatic and renal GSH were observed only at the highest level of exposure. No reductions in tissue AA were observed in either species at any exposure level. In both species, blood levels of GSH and AA remained unchanged following exposure. Mainstream smoke (240 puffs) from flue-cured or dark, air-cured tobaccos elicited a significant, immediate reduction in pulmonary and renal GSH, but MS from low tar, filter cigarettes was without effect. Within 3 h of exposure, GSH in all tissues has returned to pre-exposure levels. Whole-body, chamber exposure to concentrated SS, generated from smouldering cigarettes, caused a dose-dependent reduction in rat pulmonary, hepatic, renal, cardiac and bladder muscle GSH but only affected pulmonary GSH in the guinea pig. Lesser effects were observed in tissues of rats exposed to diluted SS. In the rat, a comparison of the results of diethylmaleate- and smoke-induced depletion of tissue GSH suggested that, even at exceptionally high levels of exposure, there was a significant store of GSH in tissues that did not interact with tobacco smoke.