Menadione toxicity in isolated rat hepatocytes was mitigated by the antioxidant 4b,5,9b,10-tetrahydroindeno[1,2-b]indole at low concentrations (less than 100 microM), but not at high concentrations (greater than 200 microM) of menadione. When hepatocytes were incubated with menadione, there was a time-dependent and concentration-dependent inhibition of lipid peroxidation in intact cells, as well as an increase in the antioxidative potency of acetone extracts, suggesting that metabolites of menadione could inhibit oxidative stress, and that at high menadione concentrations a different mechanism was involved in cytotoxicity. A possible mechanism was suggested by the ability of acetone extracts from hepatocytes that had been incubated with menadione to increase osmotic fragility in red blood cells. This increase correlated with an increase in membrane fluidity in red blood cells, determined by flourescence polarization using the membrane probe 1,6-diphenyl-1,3,5-hexatriene. At 200 microM menadione, an increase in membrane fluidity was also observed in hepatocytes. The thiol dithiothreitol protected hepatocytes from 50 microM menadione toxicity, but not from greater than or equal to 100 microM menadione. The results suggest that while oxidative stress and arylation may be the critical mechanisms of toxicity at low menadione concentrations, at higher concentrations another mechanism such as enhanced membrane fluidity is operative.