Sphingolipids have been suggested to act as second messengers for an array of cellular signaling activities in plant cells, including stress responses and programmed cell death (PCD). However, the mechanisms underpinning these processes are not well understood. Here, we report that an Arabidopsis mutant, fumonisin B1 resistant 11-1 (fbr 11-1), which fails to generate reactive oxygen intermediates (ROIs), is incapable of initiating PCD when the mutant is challenged by fumonisin B(1) (FB(1)), a specific inhibitor of ceramide synthase. Molecular analysis indicated that FBR11 encodes a long-chain base 1 (LCB1) subunit of serine palmitoyltransferase (SPT), which catalyzes the first rate-limiting step of de novo sphingolipid synthesis. Mass spectrometric analysis of the sphingolipid concentrations revealed that whereas the fbr 11-1 mutation did not affect basal levels of sphingoid bases, the mutant showed attenuated formation of sphingoid bases in response to FB(1). By a direct feeding experiment, we show that the free sphingoid bases dihydrosphingosine, phytosphingosine and sphingosine efficiently induce ROI generation followed by cell death. Conversely, ROI generation and cell death induced by dihydrosphingosine were specifically blocked by its phosphorylated form dihydrosphingosine-1-phosphate in a dose-dependent manner, suggesting that the maintenance of homeostasis between a free sphingoid base and its phosphorylated derivative is critical to determining the cell fate. Because alterations of the sphingolipid level occur prior to the ROI production, we propose that the free sphingoid bases are involved in the control of PCD in Arabidopsis, presumably through the regulation of the ROI level upon receiving different developmental or environmental cues.