Our previous study suggested that menadione (MEN) is cytotoxic to platelets of rats due to oxidative stress. To elucidate the mechanism of this toxicity, MEN-induced depletion of total cellular thiols or depletion of gutathione was studied in intact rat platelets. Treatment with MEN resulted in dose and time dependent damage to plasma (assessed by lactate dehydrogenase leakage) and intracellular (assessed by serotonin release) membranes. These events were well correlated with total cellular thiol depletion by MEN treatment, however MEN-induced glutathione depletion occurred rapidly and before plasma and intracellular membrane damage. Unlike hepatocytes, oxidized glutathione was not formed, suggesting that protein arylation, rather than oxidative stress, might be the ultimate mechanism for MEN cytotoxicity in platelets. Platelets were pretreated with dithiothreitol to supplement the total cellular thiol pool or with 1-chloro-2,4-dinitrobenzene to deplete glutathione, a soluble thiol. Dithiothreitol pretreatment to platelets protected against MEN-induced toxicity to both plasma membranes (prevented lactate dehydrogenase leakage and changes in platelet turbidity) and intracellular membranes (prevented serotonin release), although 1-chloro-2,4-dinitrobenzene pretreatment potentiated these toxicities. These results indicate that the total cellular pool of thiols plays a vital role in maintaining plasma and intracellular membrane integrity.