2,3-Dimethoxy-5-methyl- p -benzoquinone is a common chemical structure of coenzyme Q (CoQ) that conjugates different lengths of an isoprenoid side chain at the 6-position of the p -benzoquinone ring. In a series of studies to explore the cytotoxic mechanism of CoQ homologues with a short isoprenoid side chain, we found that a CoQ analogue without an isoprenoid side chain, CoQ₀, showed marked toxicity against HeLa cells in comparison with cytotoxic homologues. Therefore, we examined the cytotoxic mechanism of CoQ₀. Different from the cytotoxic CoQ homologues that induced apoptosis, 100 µM CoQ₀ induced necrosis of HeLa cells. The CoQ₀-induced cell death was accompanied by a decrease in endogenous non-protein and protein-associated sulfhydryl (SH)-groups, but this improved with the concomitant addition of compounds with SH-groups but not antioxidants without SH-groups. In addition, UV-spectrum analysis suggested that CoQ₀ could rapidly form S-conjugated adducts with compounds with SH-groups by Michael addition. On the other hand, enzyme activities of both glyceraldehyde-3-phosphate dehydrogenase, which has a Cys residue in the active site, and α-ketoglutarate dehydrogenase complex, which requires cofactors with SH-groups, CoA and protein-bound α-lipoic acid, and CoA and ATP contents in the cells were significantly decreased by the addition of CoQ₀ but not CoQ₁. Furthermore, the decrease of an endogenous antioxidant, glutathione (GSH), by CoQ₀ treatment was much greater than the predicted increase of endogenous GSH disulfide. These results suggest that CoQ₀ rapidly forms S-conjugate adducts with these endogenous non-protein and protein-associated SH-groups of HeLa cells, which disrupts carbohydrate metabolism followed by intracellular ATP depletion and necrotic cell death.