A novel nucleoside analog, 3'-fluoro-3'-deoxyadenosine (3'F3'dAdo), was evaluated for antiviral activity against several arthropod-borne and arenaviruses in Vero cell culture. The following 50% inhibitory concentrations (EC50) of virus plaque formation were obtained against the test viruses: Semliki Forest (10.3 microM) and Venezuelan equine encephalitis (5.3 microM) alphaviruses, lymphocytic choriomeningitis (7.7 microM) and Pichinde (greater than 32 microM) arenaviruses, Punta Toro (greater than 32 microM) and San Angelo (1.6 microM) bunyaviruses, banzi flavivirus (4.0 microM), and Colorado tick fever orbivirus (0.6 microM). By comparison, the broad-spectrum antiviral agent ribavirin was active against lymphocytic choriomeningitis (18 microM), Pichinde (24 microM), Punta Toro (114 microM), and San Angelo (99 microM) viruses, but was less active against the other 4 viruses (greater than 200 microM). Vero cell proliferation and thymidine and uridine incorporation into replicating Vero cells were inhibited by 50% with 3'F3'dAdo concentrations of 36, 45, and 32 microM, respectively. In virus yield reduction assays, increasing the multiplicity of infections of Semliki Forest and Venezuelan equine encephalitis viruses reduced the inhibitory activity of 3'F3'dAdo. Using the same assay, 3'F3'dAdo was found to enhance Punta Toro virus replication up to 5-fold relative to the untreated control. By adding the nucleoside transport inhibitor nitrobenzylthioinosine (100 microM) to the culture medium, antiviral activity against the two alphaviruses was eliminated, indicating that 3'F3'dAdo uses the nucleoside transport system for cell entry. When actinomycin D (5 microM) was used to greatly suppress cellular RNA synthesis in Semliki Forest virus-infected and uninfected cells, 3'F3'dAdo preferentially inhibited viral RNA synthesis. The results of these studies indicate 3'F3'dAdo is a selective inhibitor of most of the viruses tested and should be a promising candidate for in vivo evaluations.