Splenocytes isolated from Walker 256 tumor bearing rats (Tbr) presented higher proliferation than control (Ctr) splenocytes. When incubated in the presence of the prooxidant terc-butyl hydroperoxyde (t-BOOH; 500 µM) or the chemoterapic doxorubicin (100 µM), Ctr and Tbr lymphocytes presented significant decrease in both proliferation and viability that were prevented by the antioxidant desferal. Interestingly, the effects of t-BOOH and doxorubicin were more pronounced in Tbr splenocytes and were mediated predominantly by necrotic and apoptotic cell death, respectively. Chelation of intracellular Ca²⁺ with BAPTA protected Ctr and Tbr splenocytes from t-BOOH-induced decrease in proliferation and viability, suggesting that the higher cytosolic free Ca²⁺ in Tbr splenocytes was responsible for the increased susceptibility to t-BOOH. Cyclosporin A, but not FK 506, protected both Ctr and Tbr splenocytes against t-BOOH. This indicates that the toxic effects of t-BOOH were mediated by mitochondrial permeability transition. Indeed, incubation of Tbr splenocytes in the presence of t-BOOH resulted in a cyclosporin A-sensitive decrease in mitochondrial membrane potential. In conclusion, increased cytosolic free Ca²⁺ concentrations renders these cells more susceptible to mitochondrial permeability transition and death. This may explain the process of lymphopenia observed during chemotherapy-associated oxidative stress, such as that induced by the antineoplastic agents anthracyclines and camptothecins.