Culex annulirostris mosquitoes infected with SFV-S, a temperature-sensitive, small-plaque mutant of Semliki Forest virus isolated from cultured Aedes albopictus cells, could not be infected later with wild-type virus (SFV-W). Interference occurred as soon as 24 h after infection with SFV-S. In mosquitoes infected with a mixture of the two viruses, both replicated normally.