In this study the effect of metabolism of menadione (2-methyl-1,4-naphthoquinone) on ATP generation in isolated rat hepatocytes was investigated. Menadione-induced cytotoxicity correlated well with the depletion of ATP. Loss of viability lagged approximately 25 min behind the depletion of ATP. Our results suggest that depletion of ATP may be mediated by interference with glycolysis and protein breakdown, resulting in a lack of oxidizable substrates for ATP generation. (i) Menadione reduced proteolysis to 27% of control after 60 min of incubation. (ii) Increased glycogenolysis was not accompanied by accumulation of glycolytic end-products. The increased levels of glucose 6-phosphate were mainly metabolized to glucose. (iii) Menadione induced a time- and concentration-dependent inhibition of the glyceraldehyde-3-phosphate dehydrogenase activity, although no accumulation of glycolytic intermediates was found. The data presented suggest that glycolysis may be inhibited upstream of glyceraldehyde-3-phosphate dehydrogenase. (iv) Suppletion of metabolic substrates (pyruvate, oxaloacetate, and glutamine) postponed the menadione-induced ATP depletion and delayed the onset of cell killing. The protecting effect of these metabolic substrates could be reversed by atractyloside, an inhibitor of the ADP/ATP translocase. The temporary protection of metabolic substrates suggests that additional mechanisms (e.g., cofactor depletion, mitochondrial damage, enzyme inactivation) may play a role in menadione-induced ATP depletion. The present study substantiates the critical role of ATP depletion in menadione-induced cell death.