Previously, we have shown that ceramide is able to directly bind to and activate c-Raf and to trigger the downstream classical mitogen-activated protein kinase (MAPK/ERK) cascade in glomerular mesangial cells [Proc. Natl. Acad. Sci. USA 93 (1996) 6959]. In this study, we show that ceramide acts differently in glomerular endothelial cells in that treatment of endothelial cells with exogenous ceramide leads to a potent activation of the stress-activated protein kinase (SAPK/JNK) cascade but not to an activation of the classical ERK cascade. A similar effect was observed with the inflammatory cytokines TNFalpha and IL-1beta, which activate a sphingomyelinase and thereby increase intracellular ceramide levels. The activation of JNKs as shown by c-Jun phosphorylation assays was paralleled by increased phosphorylation of the two JNK isoforms, p45 and p54. In addition, also the activator of JNKs, SEK1, was found to be increasingly phosphorylated by exogenous ceramide as well as by TNFalpha. In contrast, dihydroceramide had no effect on JNK or SEK1 phosphorylation. To see whether ceramide directly binds to MEKK1, which is the c-Raf analog in the SAPK cascade, a radioiodinated photoaffinity labeling analogue of ceramide, (N-[3-[[[2-(125I)iodo-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl]oxy]-carbonyl] propanoyl]-D-erythro-sphingosine) ([125I]TID-ceramide) was used. Stimulation of endothelial cells with this [125I]TID-ceramide for 5 min followed by a short photolysis defined MEKK1 as a direct target of ceramide. With the same method, protein kinase C-alpha (PKC-alpha) was identified as a ceramide target. In contrast, no binding to c-Raf or the MEKK1 activator p65-PAK could be detected. A direct binding of ceramide to MEKK1 was also confirmed by affinity chromatography using a ceramide-coupled sepharose column. Furthermore, the ceramide-activated SAPK/JNK cascade is clearly involved in the mechanism of apoptosis, since in the presence of a JNK inhibitor, ceramide-induced DNA fragmentation is significantly reduced. In summary, we have shown that ceramide potently activates the SAPK cascade but not the ERK cascade in endothelial cells, which contrasts to mesangial cells where ceramide activates the ERK pathway and has only a minor effect on the SAPK cascade. Regarding the direct target of ceramide binding and action in endothelial cells, we identified MEKK1 as a further member of the growing family of ceramide-activated protein kinases.