Flavivirus-induced polykaryocytes were detected in monolayers of Aedes albopictus (clone C6/36) mosquito cells as early as 20 min after adsorbing virus to these cells. A high multiplicity of infection with dengue (DEN)-1, 2, 3, 4, Japanese encephalitis, and yellow fever viruses was required to demonstrate fusion from without (FFWO) with these flaviviruses. Optimal conditions for FFWO included exposure of adsorbed virus to pH 6.0 and an incubation temperature of 39 degrees C. DEN-2 monoclonal antibodies to the envelope E glycoprotein inhibited cell fusion, whereas monoclonal antibodies to the prM and NS1 proteins did not inhibit cell fusion. These results indicate that flaviviruses cause FFWO soon after adsorption to C6/36 mosquito cells and the process is most likely mediated by the virion envelope E glycoprotein.