It has been found previously that hydroxycobalamine (vitamin B 12b ) amplifies significantly the cytotoxic effect of ascorbic acid (vitamin C) added to cells for а long period of time (48 h). However, according to pharmacokinetics, the concentration of vitamin C in vivo decreases to a physiological value within a short period of time (2–3 h) after the injection. Therefore, in this study we examined the cytotoxic effect of a short-time (up to 2 h) exposure of human larynx carcinoma HEp-2 cells to a combination of vitamins B 12b and C (B 12b +C). The kinetics of the B 12b +C-caused extracellular oxidative burst in this time interval was also explored. Vitamin B 12b combined with ascorbic acid provoked a rapid accumulation of extracellular hydrogen peroxide followed by intracellular oxidative stress, DNA single-strand breaks, and the initiation of apoptosis. The chelators of iron phenanthroline and desferrioxamine prevented B 12b +C-induced DNA single-strand breaks and cell death but not the accumulation of H 2 O 2 in culture medium. The nonthiol antioxidants pyruvate and catalase were effective in preventing the prooxidant and cytotoxic effects of B 12b +C. Thiols, when added simultaneously with the combined vitamins, inhibited these effects only partially (N-acetylcysteine, GSH) or even amplified them (dithiothreitol). The results obtained point to the determining role of oxidative burst and iron-dependent DNA damage in the cytotoxic effect of short-time exposure to B 12b +C combination.