Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients and human immunodeficiency virus (HIV)-infected homosexual males. We evaluated the cytotoxic effects of heat shock protein 90 (HSP90) inhibitors on PEL cells. The HSP90 inhibitors geldanamycin (GA), 17(allylamino)-17-demethoxygeldanamycin (17-AAG), and radicicol dramatically inhibited cell proliferation and induced apoptosis of PEL cells through caspase activation. Furthermore, GA induced the stabilization of inhibitor of κB (IκB)α and reduced the phosphorylation of IκBα in PEL cells. HSP90 inhibitors suppressed the transcriptional activity of nuclear factor-kappa B (NF-κB) in PEL cells. It is known that the constitutive activation of NF-κB signaling is essential for the survival of PEL cells and HSP90 contributes to promote activation of NF-κB signaling. The suppression of NF-κB signaling by HSP90 inhibitors may contribute to the induction of apoptosis in PEL cells. In addition, HSP90 activity is required for KSHV replication in KSHV latently infected PEL cells. GA, 17-AAG and radicicol reduced the production of progeny virus from PEL cells at low concentrations, which do not affect PEL cell growth. Our results suggest that HSP90 activity is required for both the survival of PEL cells and viral replication in PEL cells, and that pharmacologic inhibition of HSP90 may be an effective treatment for PEL and KSHV-related diseases.