To clarify the role of electrostatic interactions in the binding of Sindbis virus (SNV) to cell membrane receptors, we investigated the effect of different polyions on the initial steps of infection of Vero cells by the virus. Several polyanions (mucin, heparin, polygalacturonic acid) and polycations (polylysine, protamine, polybrene) were able to reduce the replication of SNV when present in the viral adsorption period, whereas others (chondroitin sulfate, polymyxin B sulfate, histone) were devoid of any activity. Therefore the electric charge alone is not sufficient to explain the action of compounds. The effects of polyions on receptor binding, on bound virus, and on internalized virus have been examined. All the drugs inhibited SNV infection by affecting its binding to the cellular receptor. The results indicated that heparin and mucin act directly on the virus particle while polycations bind to the cell membrane receptor for the virus, protamine being effective on both targets. Since among polyanions glycosaminoglycans showed a strong inhibiting activity, the involvement of these molecules in the virus surface receptor was assessed by enzyme digestion of cell membrane with heparinase and chondroitin ABC lyase.