The general objective of this study was to examine the role of oxidative stress and transition metal catalysis in lipofuscinogenesis in postmitotic cells. Effects of 30 microM ferric chloride and different concentrations of desferrioxamine, ranging from 12.5 to 50 microM, on lipofuscin content were examined in rat cardiac myocytes at 6 and 12 days of in vitro age, under 20% and 40% ambient oxygen concentration. Lipofuscin was quantified by microspectrofluorometry of individual cells. Using X-ray microanalysis, iron was found to be sequestered in the secondary lysosomes that also contain the fluorescent lipofuscin material. Augmentation of iron in the culture medium markedly increased the level of lipofuscin accumulation while desferrioxamine had the opposite effect. Both of these effects were more pronounced at higher oxygen tension. Iron and oxygen had a cumulative effect on lipofuscin accumulation. Results are interpreted to indicate that oxidative stress and iron, in loosely bound form, are causal factors in lipofuscinogenesis. The possibility of employing microspectrofluorometry of lipofuscin as an indicator of oxidative stress is emphasized.