The effects of hydrogen peroxide on cell viability and, in particular, on lysosomal integrity were investigated in a model system of cultured, established, macrophage-like J-774 cells. The cells were found to rapidly degrade added hydrogen peroxide, withstanding concentrations < or = 250 microM without cell death; however, all tested concentrations (100-500 microM) substantially decreased cellular ATP to approximately the same degree. Concentrations of hydrogen peroxide > or = 500 microM resulted in a pronounced and rapid decrease in cell viability preceded by the loss of lysosomal integrity, as judged by the relocalization of acridine orange, a lysosomotropic weak base, in pre-labelled cells. Hydrogen peroxide-induced relocalization of acridine orange and cell death were either enhanced or much prevented, according to if the cells were initially allowed to endocytose ferric iron or the specific iron-chelator deferoxamine, respectively. Depletion of ATP, however, was not associated with the loss of lysosomal integrity and viability regardless of iron or deferoxamine pretreatment. Pre-exposure to E-64, an inhibitor of lysosomal thiol proteases, resulted in the reduction of both lysosomal membrane damage and cell death. The results are interpreted as indicating (i) generation of hydroxyl radicals within the secondary lysosomal compartment due to the occurrence of reactive ferrous iron, leading to (ii) peroxidative alterations of the lysosomal membrane resulting in (iii) loss of lysosomal membrane integrity with dissipation of the proton gradient and leakage of lysosomal contents, including hydrolytic enzymes, into the cell sap.