Geldanamycin (GA) is an antibiotic targeting the ADP/ATP binding site of heat shock protein 90 (Hsp90). In screening for anti-herpes simplex virus type 1 (HSV-1) candidates, we found GA active against HSV-1. HSV-1 replication in vitro was significantly inhibited by GA with an 50% inhibitory concentration of 0.093 microM and a concentration that inhibited cellular growth 50% in comparison with the results seen with untreated controls of 350 microM. The therapeutic index of GA was over 3700 (comparable to the results seen with acyclovir). GA did not inhibit HSV-1 thymidine kinase. Cells infected with HSV-1 demonstrated cell cycle arrest at the G(1)/S transition; however, treatment with GA resulted in a cell cycle distribution pattern identical to that of untreated cells, indicating a restoration of cell growth in HSV-1-infected cells by GA treatment. Accordingly, HSV-1 DNA synthesis was suppressed in HSV-1(+) cells treated with GA. The antiviral mechanism of GA appears to be associated with Hsp90 inactivation and cell cycle restoration, which indicates that GA exhibits broad-spectrum antiviral activity. Indeed, GA exhibited activities in vitro against other viruses, including severe acute respiratory syndrome coronavirus. Since GA inhibits HSV-1 through a cellular mechanism unique among HSV-1 agents, we consider it a new candidate agent for HSV-1.