G SH plays an important role in cellular defense against a wide variety of toxic electrophiles via the formation of thioether conjugates. We studied the role of GSH in murine tumor cell defense against a novel class of sulfhydryl-reactive antineoplastics, the sesquiterpene lactones (SL). Incubation of P815 mastocytoma cells with any of the four SL tested (vernolepin, helenalin, elephantopin, and eriofertopin) for 1 h resulted in 70-97% depletion of GSH. The importance of GSH resynthesis upon exposure of tumor cells to SL was evaluated with the use of buthionine sulfoximine (BSO), a selective, nontoxic inhibitor of gamma-glutamylcysteine synthetase. Inhibition of GSH synthesis with 0.2 mM BSO markedly enhanced SL-mediated cytolysis of four murine tumor cell lines. A 6- to 34-fold reduction in the amount of SL causing 50% lysis was obtained with BSO. Addition of BSO to P815cells either during or immediately after a 1-h pulse with 10 micrograms/ml of vernolepin increased cytolysis from less than 3% to 78-82%. However, a 1.5-h delay in the addition of BSO to such cells, which allowed for substantial resynthesis of GSH, reduced cytolysis to 30%. Recovery of GSH synthetic capacity after BSO treatment correlated with loss of the synergistic effect of BSO on lysis by vernolepin. BSO did not augment cytolysis by six other antineoplastics (doxorubicin, mitomycin C, vinblastine, cytosine arabinoside, maytansine, and 1,3-bis-[2-chloroethyl]-1-nitrosourea [BCNU]). Of these, only BCNU depleted cellular GSH. Lysis by jatrophone, another GSH-depleting antitumor agent, was increased 21-fold by BSO. Since prolonged incubation with BSO alone results in near-complete GSH depletion without loss of cell viability, SL-mediated cytolysis is probably not a result of GSH depletion. We have demonstrated, however, a critical role for GSH synthetic capacity as a determinant of tumor cell susceptibility to cytolysis by SL. GSH also plays an important role in cellular defense against oxidative injury. Vernolepin, acting as a GSH-depleting agent, markedly sensitized tumor cells to lysis by H2O2 (greater than 6.5-fold increase with 20 micrograms/ml of vernolepin). These findings suggest the possibility that the coordinated deployment of sulfhydryl-reactive antitumor agents, BSO, and oxidative injury might constitute an effective chemotherapeutic strategy.