The objective of this study was to test further the hypothesis that oxidative stress is a major causal factor in lipofuscin formation. We have previously shown that cultured cardiac myocytes constitute a suitable model system for the study of factors influencing lipofuscinogenesis. The specific aim of the present study was to elucidate the effects of the chain-breaking free radical scavenger alpha-tocopherol, and the chelators desferrioxamine, EDTA and DTPA on the accumulation of lipofuscin. The effects were examined at different degrees of oxidative stress, obtained by varying the ambient oxygen concentration from 5 to 40%. Lipofuscin was quantified by microspectrofluorometry. Lipofuscin-specific, yellow autofluorescence increased with time in culture, and with enhanced oxidative stress. Increasing concentration of alpha-tocopherol, up to 40 microM, had an inhibitory effect on lipofuscin accumulation that was most pronounced at high oxidative stress. Desferrioxamine and DTPA, both caused a pronounced reduction in lipofuscin formation, while EDTA had no significant effect. The findings are interpreted to support the concept that oxidative stress is a causal factor in lipofuscinogenesis, and that lipofuscin is a product of autophagocytosed, membrane-rich material subjected to free radical-induced, metal-catalyzed peroxidation, fragmentation, and polymerization within the lysosomal vacuome.