In a previous investigation, we demonstrated that after CD95/Fas triggering, raft-associated GD3 ganglioside, normally localized at the plasma membrane of T cells, can be detected in mitochondria, where they contribute to apoptogenic events. Here, we show the association of the glycosphingolipid GD3 with microtubular cytoskeleton at very early time points following Fas ligation. This was assessed by different methodological approaches, including fluorescence resonance energy transfer, immunoelectron microscopy, and coimmunoprecipitation. Furthermore, docking analysis also showed that GD3 has a high affinity for the pore formed by 4 tubulin heterodimers ( type I pore), thus suggesting a possible direct interaction between tubulin and GD3. Finally, time-course analyses indicated that the relocalization of GD3 to the mitochondria was time related with the alterations of the mitochondrial membrane potential. Hence, microtubules could act as tracks for ganglioside redistribution following apoptotic stimulation, possibly contributing to the mitochondrial alterations leading to cell death.-Sorice, M., Matarrese, P., Tinari, A., Giammarioli, A. M., Garofalo, T., Manganelli, V., Ciarlo, L., Gambardella, L., Maccari, G., Botta, M., Misasi, R., Malorni, W. Raft component GD3 associates with tubulin following CD95/Fas ligation. FASEB J. 23, 3298-3308 ( 2009). www.fasebj.org