Differential changes in various sphingolipids between TNF-induced necroptosis and apoptosis were investigated using liquid chromatography-tandem mass spectrometry. A marked increase in d18:1/16:0 ceramide was detected in U937 cells treated with TNF in the presence of Z-VAD-fmk (VAD). The level of d18:1/16:0 ceramide in necroptosis was almost twice as high as that in apoptosis after 4h, while an increase in PI-positive cells was observed only in necroptosis within 4h. Necroptosis-resistant U937 (UNR) sublines were established to more clearly discriminate between necroptosis and apoptosis. All three UNR sublines were almost completely resistant to the treatment with TNF/VAD, but were as sensitive to TNF-induced apoptosis as parental cells. The expression of RIP3, a pivotal kinase in necroptosis, was lost in all three UNR sublines. In contrast with the large increase in ceramide levels in TNF/VAD-treated parental cells, they were only slightly increased in UNR cells. Although intracellular levels of reactive oxygen species (ROS) were elevated in both necroptosis and apoptosis, the treatment with butylated hydroxyanisole, an antioxidant, significantly inhibited increases in ceramide levels and PI-positive cells only in necroptosis. These results implicate that the ROS-induced large increase in ceramide levels may play a role in plasma membrane permeabilization in TNF-induced necroptosis.