Many viruses, including flaviviruses, display affinity for cell surface heparan sulfate (HS) proteoglycans with biological relevance in virus attachment/entry. This raises the possibility of the application of HS mimetics in antiviral therapy. We have evaluated the antiviral effect of the sulfated polysaccharides, suramin, pentosan polysulfate (PPS) and PI-88, which are currently approved or in trial for clinical use, against dengue virus (DEN) and the encephalitic flaviviruses, Japanese encephalitis virus, West Nile virus, and Murray Valley encephalitis virus. A flow cytometry-based method for the measurement of inhibition of virus infectivity was developed, which showed the in vitro antiviral activity of the three compounds, albeit with differences in efficiency which were virus-dependent. The 50% effective concentration (EC(50)) values for DEN inhibition were in the order: PPS<suramin<PI-88, and for Japanese encephalitis virus, PPS<PI-88<or=suramin. Heparin inhibited the DEN infectivity 30-fold more efficiently than the best of the test compounds, which was not the case for encephalitic flaviviruses. The in vitro anti-flaviviral effectiveness of the HS mimetics did not reliably predict their in vivo therapeutic activity. In mouse models for DEN and flaviviral encephalitis, only PI-88 demonstrated a significant beneficial effect in disease outcome.