Ascorbic acid (AA) was found to be cytotoxic to neuroblastoma cells in vitro and in vivo. In the present, study we investigated whether the reduced--(AA) or oxidized form (dehydroascorbic acid, DhAA) and its rapidly formed metabolites were the main cytotoxic agents. In neuroblastoma SK-N-SH cells, AA was found to be more cytotoxic than DhAA, although considerably higher amounts of [14C]DhAA than of [14C]AA were incorporated. In contrast, SK-N-LO cells derived from neuroectodermal tissue in fact showed a similar uptake but were much less injured by both substances. We observed that uptake of [14C]AA and [14C]DhAA was impaired in the presence of dithiothreitol and glutathione. Once inside the cell, [14C]DhAA was partially reduced to [14C]AA. From these data we conclude first that at least part of AA is oxidized prior to its uptake, and second that the reduced form of AA and perhaps ascorbyl radicals but not DhAA or its metabolites are the most important forms in mediating cytotoxic reactions in neuroblastoma cells. Furthermore, the results strengthen the previous assumption that AA acts as a pro-oxidant in neuroblastoma cells and supports its use in the treatment of neuroblastoma, especially in combination with existing chemotherapeutics.