The effect of geldanamycin (GA), a specific inhibitor of heat shock protein 90 (Hsp90), on gene expression and replication of human cytomegalovirus (HCMV) was studied in human embryonic lung (HEL) fibroblasts. Kinetic analysis indicated that GA delayed synthesis of major immediate early (MIE), early and late viral proteins, and blocked a second tier of the synthesis of these proteins that occurred in untreated cells after 48 h post-infection (pi). Moreover, when HCMV-infected HEL cells were maintained with medium containing 40 nM GA for 6 days, with medium changes at 2-day intervals, the virus yield was reduced to an undetectable level. On a molecular level, the cellular kinase Akt and the transcription factor NFkappaB were activated in HCMV-infected cells within 30 min pi. NFkappaB was shown to be essential for MIE gene expression. However, in GA-treated cells, activation of both Akt and NFkappaB was greatly inhibited. Because LY294002, an inhibitor of cellular phosphatidylinositol 3-kinase (PI3-K), also prohibited HCMV-mediated activation of Akt and NFkappaB and synthesis of the MIE proteins, PI3-K signalling was necessary for expressing the MIE genes. These results suggest that the inhibitory effect of GA on HCMV replication is primarily caused by the disruption of the PI3-K signalling pathway, leading to the activation of NFkappaB, which plays a crucial role in expression of the critical MIE genes.