Mitogen-activated protein (MAP) kinase activity is essential for tumor necrosis factor (TNF) α receptor 1 regulation of intestinal epithelial cell proliferation. However, the mechanism of TNF-αmediated activation of extracellular signal-regulated kinase (ERK)/MAP kinase has not been established clearly. Both TNF-α and cell-permeable ceramide have been reported to increase the kinase activity of kinase suppressor of Ras (KSR). To determine the role of KSR in TNF-α-induced ERK1/ERK2 activation, we studied young adult mouse colon cells expressing a dominant-negative, kinase-inactive(ki) KSR. We report that TNF-α, a cell-permeable ceramide, and sphingomyelinase stimulate ERK1/ERK2 activation and increase the phosphoserine content of KSR, which are inhibited by kiKSR expression in intact cells. Furthermore, TNF-α-induced Raf-1 threonine phosphorylation, kinase activity toward MEK1, and association with KSR are also inhibited by kiKSR expression. Our data also show by sequential in vitro kinase assays that TNF-α enhances KSR phosphorylation of Raf-1 on threonine, enhancing Raf-1 kinase activity toward MAP kinase kinase. We therefore conclude that KSR is an essential upstream regulator of TNF-α-stimulated ERK1/ERK2 activation, most likely mediated via direct phosphorylation of Raf-1.