Calcitriol, the hormonal form of vitamin D, enhances the anticancer activity of the immune cytokine tumor necrosis factor, interleukin 1 and interleukin 6 in human breast and renal cell carcinoma cells without affecting the cytotoxic action of interferon-alpha or killer lymphocytes. It also enhances cytotoxicity induced by the anticancer drug doxorubicin, by the redox cycling quinone menadione, and by the reactive oxygen species hydrogen peroxide. The synergistic interaction was accompanied by increased oxidative stress, as manifested by glutathione depletion and was abolished by exposure to the thiol antioxidant N-acetylcysteine. The hormone on its own brought about an increase in the cellular redox state as reflected in the ratio between oxidized and reduced glutathione and glyceraldehyde-3-phosphate dehydrogenase, and a reduction in the expression of the antioxidant enzyme Cu/Zn superoxide dismutase. These results support the notion that the interplay between active vitamin D derivatives and other anticancer agents such as immune cytokines and anticancer drugs plays a role in the in vivo anticancer activity of vitamin D and that reactive oxygen species are involved in the anticancer activity of vitamin D on its own and in its cross-talk with other anticancer modalities.