Conventional chemotherapy is the golden standard for therapy of Hodgkin Lymphoma (HL). Nevertheless, considerable toxicity and secondary malignancies indicate the need for targeted therapy that preferentially kills the malignant cells. The molecular chaperone heat shock protein 90 (HSP90) is expressed in all mammalian cells, but it is overexpressed in malignancy. 17-AAG, a small molecule inhibitor of HSP90, has been shown to induce apoptosis and cell cycle arrest in a variety of tumor types. In the present study we show that HSP90 is overexpressed in the primary Hodgkin and Reed-Sternberg (HRS) cells, as well as in HL derived cells lines. Inhibition of HSP90 17-AAG showed antiproliferative effect in HL derived cell lines in a dose dependent manner. Cell death was due to apoptosis, as determined by Annexin-V staining and FACS analysis. Apoptosis was mediated by the activation of the caspase pathway, especially by caspase 8, 9, and 3. Inhibition of caspase activity by the pancaspase inhibitor Z-VAD-FMK partially reversed the 17-AAG lethal effect. 17-AAG had no significant on the level of the antiapoptotic Bcl-2 family members or the cellular or X-Linked inhibitors of apoptosis. In contrast, there was considerable degradation of cFLIP. Moreover, 17-AAG treatment reduced the intracellular levels of molecules that have been shown to be of key importance in HRS cell survival and proliferation, including AKT and the phosphorylated ERK1/2, but with minimal change in total ERK1/2. Cell cycle arrest was observed at G0/G1 or at G2/M phase, and was mediated by reduction in the levels of MDM2, cyclin D1 with cdk4 and cdk6, and cyclin B1. The potential synergy of 17-AAG with conventional chemotherapy and anti-TRAIL death receptor monoclonal antibody, was explored by the simultaneous incubation of HL derived cells with both doxorubicin or antibodies against TRAIL receptors R1 and R2, respectively. The combination of 17-AAG with doxorubicin or anti-TRAIL antibodies was significantly more effective than either agent alone. Based on these data we are conducting a phase II study of 17-AAG in patients with relapsed classical HL.