The pro-apoptotic TNF-family ligand TRAIL is currently one of the most promising novel anti-cancer agents. Although TRAIL has been intensively studied in the past decade, the signalling cascades which lead to and regulate TRAIL-induced apoptosis are still not completely elucidated. Furthermore, since in vivo treatment with TRAIL alone did not reveal very spectacular outcomes, particular importance is given to characterizing mechanisms that may amplify TRAIL-induced apoptosis. The present study investigated the role of acid sphingomyelinase (ASM) and ceramideenriched membrane platforms for the apoptotic pathway triggered by TRAIL upon ligation of DR5 receptors. The major findings of the study are: • ASM is activated by TRAIL and plays a crucial role in apoptosis induced by physiological doses of TRAIL. • TRAIL activates the ASM via a direct or indirect redox mechanism. • TRAIL-induced ASM activation leads to generation of ceramide-enriched membrane platforms on the outer leaflet of the plasma membrane. • Ceramide-yenriched membrane platforms are essential for TRAIL-induced apoptosis. • Ceramide-enriched membrane platforms serve to trap ligated DR5 receptors leading to a massive aggregation of the receptors (receptor clustering). • Receptor clustering is essential for successful induction of apoptosis by TRAIL via amplification of the primary signal. Furthermore, the present study identifies a novel mechanism of amplification of TRAILinduced apoptosis via ceramide-enriched membrane platforms. Since ceramide platforms are able to amplify the killing potential of very low doses of TRAIL, this mechanism may prove of relevance for the in vivo treatment with TRAIL. The results demonstrate: • Non-lethal doses of doxorubicin and TRAIL have a synergistic apoptotic effect. • Non-lethal doses of doxorubicin are able to induce ceramide-enriched membrane platforms. • Doxorubicin-induced ceramide platforms are generated via activation of the ASM. • Doxorubicin-induced ceramide platforms trap and cluster the ligated DR5 receptors leading to successful induction of apoptosis.