Previous research showed that increasing membrane sphingomyelin (SPH) levels in rat pheochromocytoma (PC12) cells to the same extent as that seen in some brain regions with aging dramatically increases the vulnerability to oxidative stress (OS). These increases in vulnerability were determined by assessing deficits in the ability of these cells to extrude and/or sequester Ca2+ following 30 mM KCl-induced depolarization (recovery). The purpose of the present experiments was to discern whether increasing the levels of particular SPH metabolite(s), i.e., ceramide (Cer), sphingosine (Ssine), or sphingosine-1-phosphate (SPP), or indirectly increasing the concentrations of these metabolites with sphingomylinase (Sase), would interact with the cell's sensitivity to OS induced by low (5 microM) or high (nonlethal, 300 microM) H2O2. In addition, the OS vulnerability was examined as above under decreased SPH levels by exposing the cells to L-cycloserine (Lcc), which prevents SPH synthesis. Both Sase and SPP significantly decreased Ca2+ recovery of PC12 cells after H2O2 exposure. Conversely, Lcc-treated cells showed no further OS-induced decrements in recovery below those seen in controls. SPP significantly decreased glutathione levels (GSH) in the absence of OS. Repletion of GSH with 20 mM N-acetylcysteine significantly attenuated the effect of 5 microM H2O2 on recovery in SPP-treated cells and decreased sensitivity of SPP-treated cells to low doses of OS. Overall, our results suggest a critical role for GSH and SPP in the regulation of OS vulnerability, especially as it relates to Ca2+ homeostasis.