The role of lipid peroxidation in menadione-mediated toxicity was studied in neonatal rat cardiomyocytes. Incubation of cardiomyocytes with menadione resulted in depleted cellular glutathione levels, increased intracellular Ca2+ and increased lipid peroxidation which all occurred prior to cell degeneration. Pre-treatment of cells with cysteine suppressed the menadione-induced cell degeneration and prevented changes in glutathione levels, intracellular Ca2+, and lipid peroxidation. Pre-treatment of cells with fura-2 acetoxymethyl ester, a Ca2+ chelator, reduced menadione-induced cell degeneration and lipid peroxidation but it did not block cellular glutathione depletion. Pre-treatment of cells with deferoxamine mesylate, an iron chelator, also reduced both menadione-induced cell degeneration and lipid peroxidation; however, it did not prevent the menadione-induced increase in intracellular Ca2+, nor the depletion of glutathione. Thus, the inhibition of menadione-induced lipid peroxidation by deferoxamine mesylate prevented cell degeneration even though intracellular Ca2+ remained elevated and glutathione remained depleted. The protective effects of deferoxamine mesylate and fura-2 AM on menadione's toxicity were inhibited by addition of FeCl3 to cells. Ferric ions did not inhibit the protective effect of cysteine. These data suggest that menadione-induced cardiomyocyte degeneration is directly linked to iron-dependent lipid peroxidation and less tightly coupled to elevation in intracellular Ca2+ or depletion of glutathione.