The objective of this study was to investigate the relationship between intracellular glutathione (GSH) and lipofuscinogenesis in an established model system of cultured postmitotic neonatal rat cardiac myocytes exposed to moderate oxidative stress with respect to culture conditions (cells grown at 21% oxygen pressure). Intracellular glutathione was depleted by exposing cell cultures to buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase; this caused an increase in lipofuscin-specific autofluorescence, reflecting lipofuscin accumulation. Cell cultures exposed to 100 microM BSO exhibited the following reactions, as compared to control cells: the intracellular glutathione level decreased 78%, 86%, and 89%, and lipofuscin-specific autofluorescence increased 26%, 28%, and 77% after 5, 8, and 14 days of treatment, respectively. Compared to the glutathione levels found in newly excised heart tissue, concentrations in cultured cardiac myocytes were lower during the first few days after culture establishment, probably a result of the cell preparation technique used. Due to this effect, differences between BSO-treated cells and control cells in regard to glutathione concentration and lipofuscin accumulation were more pronounced during the remainder of the 2-week culture period. Lipid peroxidation measured as thiobarbituric acid reactive substances was not increased in BSO-treated cells. These results are in agreement with the oxidative-stress theory of lipofuscinogenesis previously proposed from the authors' laboratory. The authors show that reduced GSH level leads to a simultaneous increase in accumulation of lipofuscin in cardiac myocytes, possibly by increasing the level of cytosolic hydrogen peroxide.