Organ cultures have become an important method for the study of some of the biochemical reactions in which ascorbate takes part, and there are conflicting views on the stimulatory and inhibitory actions of ascorbate on cell growth. This study aimed to clarify the inhibitory mechanism of ascorbate using 3T6 fibroblasts. Cells were exposed to ascorbate at various concentrations in medium. The results showed that 3T6 fibroblasts were killed in medium containing more than 0.3mM ascorbate. This lethal effect of ascorbate on cells was inhibited by the addition of catalase, an enzyme that decomposes hydrogen peroxide (H₂O₂), to the medium. When the medium with ascorbate was incubated in the absence of cells, the amount of H₂O₂ generation depended on the ascorbate concentration, and decreased in inverse proportion to the serum concentration added to the medium. The addition of albumin, which is the main protein in serum, also inhibited H₂O₂ generation in the medium with ascorbate. However H₂O₂ generation was not inhibited completely by serum and albumin. These results indicate that cytotoxicity of ascor-bate is induced by H₂O₂.