To evaluate the protective role of cell glutathione (GSH) against the toxicity of cadmium, the effect of GSH depletion on the metal toxicity was investigated and the role of glutathione was compared with that of zinc-induced metallothioneins (MTs). A 6-h incubation of cultured Chinese hamster V79 cells with 0.2 mM L-buthionine-SR-sulfoximine (BSO), a selective inhibitor of gamma-glutamylcysteine synthetase, resulted in approx. 95% depletion of GSH in the cells. The depletion of GSH did not influence the rate of cell growth, the amount of cell protein or the chromosome structure during culture for at least 24 h. Cells depleted or not depleted of GSH were challenged with (1-5).10(-5) M CdCl2 for 2 h and subsequent cell growth was evaluated. The cytotoxicity of cadmium was enhanced with increasing duration of BSO pretreatment and was correlated with the decrease of cell GSH, indicating that GSH constitutes a cellular defense against toxicity by cadmium. Inducibility of MTs by zinc was investigated in cultured V79 cells. Incubation of the cells with 1.10(-4) M zinc acetate did not result in accumulation of MTs over the control values for up to 2 h. Thereafter, however, the synthesis of MTs increased with increasing duration of zinc treatment and an approx. 9-fold increase in the amount of MTs was observed 10 h after addition of zinc. Depletion of cell GSH by BSO did not much influence the increased accumulation of MTs by zinc. In contrast, zinc at the same concentration did not influence the level of cell glutathione up to 12 h. The cytotoxicity of cadmium was markedly reduced in the cells pretreated with zinc and the protective effect of zinc was dependent upon duration of pretreatment, being parallel with the increased accumulation of MTs. Protection of cells from cadmium toxicity by zinc pretreatment was as or a little more effective in the cells depleted of GSH as in those not depleted. Thus, glutathione appears to be an intrinsic protector against cadmium toxicity, while MTs serve as an induced cellular defense that is mobilized against heavy metal stress, but takes more than 2 h to accumulate in significant amounts. Accordingly, it is suggested that GSH and MTs have cooperative protective roles against cadmium toxicity, as an initial defense for the former and a second-stage defense for the latter.