Ribavirin, interferon-alpha (IFN-alpha), 6-azauridine and glycyrrhizin were tested in vitro for their antiviral activities against 11 pathogenic flaviviruses belonging to principal antigenic complexes or individual serogroups of medical importance: dengue, Japanese encephalitis, mammalian tick-borne and yellow fever virus (YFV) groups. Antiviral activity was estimated by the reduction of the cytopathic effect of each flavivirus in Vero cells and by the reduction in virus titer. Cytotoxicity was evaluated by determining the inhibition of Trypan blue exclusion in confluent cell cultures and by the evaluation of the inhibitory effect on cell growth. The specificity of action of each tested compound was estimated by the selectivity index (CC(50)/EC(50)). IFN-alpha proved to be a selective and potent inhibitor of the replication of the 11 tested pathogenic flaviviruses. Ribavirin and 6-azauridine proved to be active on the replication of the 11 tested pathogenic flaviviruses at the concentrations which did not alter normal cell morphology, but they were not selective inhibitors when selectivity indices were evaluated with regard to the inhibition of cell growth because of their cytostatic effect. Glycyrrhizin inhibited the replication of flaviviruses at high non-cytotoxic concentrations. These antiflavivirus compounds should be further evaluated for their efficacy in the treatment of flavivirus infections in vivo.