The anticancer potential of ascorbic acid (AA) has been controversially discussed for decades. Although the cytotoxic effect of pharmacologic concentrations of ascorbic acid has already been successfully demonstrated in numerous studies in vitro, it could not be verified to the same extent in vivo. We propose that the ubiquitous metabolite pyruvate diminishes the effect of AA by reacting with its presumable cytotoxic mediator hydrogen peroxide (H2O2). MIT assays confirm that co-incubation with 1.4 mM pyruvate abolishes the cytotoxic effect of pharmacologic concentrations of AA in all cancer cell lines tested (human melanoma (WM451-Lu), breast (MCF-7) and hypopharyngeal cancer cells (FaDu)). We further investigated whether pyruvate diminishes the anticancer effect of AA by interfering with the generation of H2O2. Therefore, we analyzed the concentration of AFR, a proposed intermediate in the AA-dependent formation of H2O2, by electron paramagnetic resonance spectroscopy, during incubation with AA and pyruvate in WM451-Lu cells as a model system. In addition, we measured H2O2 concentration by indirect detection with Clark-type oxygen electrode. AFR concentration was not significantly influenced by pyruvate, whereas H2O2 concentration was significantly reduced. In parallel, pyruvate concentrations of the stimulation medium declined with increasing AA and consequently H2O2 concentrations. In summary, pyruvate diminishes the cytotoxic activity of ascorbic acid in vitro. The AFR concentration measured remains unaffected by pyruvate whereas the H2O2 concentration is reduced; confirming that pyruvate directly reacts with AA-induced H2O2, without influencing its formation. However, further experiments are needed to elucidate the complex mechanisms being responsible for the reduced efficacy of AA in vivo. (C) 2017 Elsevier Inc. All rights reserved.