West Nile virus has spread rapidly throughout the United States since its introduction in 1999, into some areas that are also endemic for St. Louis encephalitis virus (SLEV). These viruses are in the same antigenic complex within the genus Flavivirus, family Flaviviridae. Further, both viruses are transmitted primarily by Culex spp. mosquitoes and use birds as amplifying hosts. These viruses could contemporaneously coinfect individual vectors wherein changes in mosquito immune responses might occur. To explore this possibility, we evaluated the effect of sequential infection with both West Nile virus and SLEV on the infection and dissemination rates of these viruses in the vector mosquito, Culex quinquefasciatus. Prior exposure to either virus lowered susceptibility to infection with the second virus, and lower dissemination rates of the second virus, compared to controls. Exposure to one virus followed by a second virus resulted in similar infection rates for the first virus to those of controls, but higher SLEV dissemination rates when exposed first to SLEV than in singly SLEV infected controls. While some mosquitoes became infected with both viruses, only one of those viruses disseminated from the midgut into the legs, indicating a midgut infection barrier to secondary infection. Lower infection rates in mosquitoes exposed to both viruses could change transmission patterns when these viruses are present at epizootic levels. © Copyright 2009, Mary Ann Liebert, Inc.