The antiviral activity of 9-(2-hydroxyethoxymethyl) guanine (Acyclovir, ACV) on the salmon herpesvirus, Oncorhynchus masou virus (OMV), was studied in vitro. ACV showed high efficacy against the fish herpesvirus OMV, Herpesvirus salmonis and channel catfish virus (CCV). Cytopathic effect (CPE) induced by 100 TCID 50/ml of OMV in rainbow trout gonad (RTG-2) cells was inhibited by 2.5 μg/ml of ACV. ACV was more effective than other compounds such as 9-β- d-arabinofuranosyladenine (Ara-A), 5-iodo-2′-deoxyuridine (IUdR) and phosphonoacetate (PA). Growth of RTG-2 cells was considerably inhibited by ACV at 25 μg/ml, but no morphological changes were observed in the cells. Replication of OMV in RTG-2 cells inoculated with 100 TCID 50/ml was completely suppressed by 2.5 μg/ml of ACV. Addition of ACV within 4 days post infection was effective in reducing OMV replication. In order to be effective, ACV had to be present continuously.