Heat shock protein 90 (Hsp90) is a survival signaling chaperone and a cancer chemotherapeutic target. However, we have found that inhibitors of Hsp90 diminished the apoptotic response induced in leukemic cells by the antitumor alkyl-lysophospholipid analog edelfosine, which acts through lipid raft reorganization. Edelfosine treatment recruited Hsp90, c-Jun N-terminal kinase (JNK) and apoptotic molecules in lipid rafts, but not the JNK regulators apoptosis signal-regulating kinase 1 (ASK1) and Daxx, or the survival signaling molecules extracellular signal-regulated kinase (ERK) and Akt. Following edelfosine treatment, Hsp90 bound to JNK in lipid rafts and Hsp90-JNK clusters were identified at the plasma membrane by immunoelectron microscopy. Hsp90 inhibition reduced JNK protein level in lipid rafts and turned proapoptotic persistent JNK activation into a transient response in edelfosine-treated cells. Decrease in edelfosine-induced JNK activation and apoptosis by Hsp90 inhibition was prevented through proteasome inhibition, suggesting that Hsp90 inhibition diminishes apoptosis by promoting JNK protein degradation. Expression of ASK1 dominant negative mutant did not affect JNK activation and apoptosis following edelfosine treatment. These data indicate that lipid raft-recruited JNK is ASK1-independent and becomes a novel Hsp90 client protein. Our results reveal a new chaperoning role of Hsp90 on JNK-mediated apoptosis following its recruitment in lipid rafts.