We recently developed an assay using the polymerase chain reaction (PCR) for the specific detection of St. Louis encephalitis (SLE) virus RNA. This assay was tested in a blind study on 7 samples of pooled mosquitoes (50 mosquitoes/pool) which were also characterized for SLE virus by plaque assay in Vero cell culture. One sample was positive for the SLE virus as determined by both the PCR assay and a combination of the plaque assay and the indirect fluorescent antibody assay. The remaining 6 samples were negative for the presence of SLE virus as determined by both methods. These data indicate that this PCR assay can be used to monitor for the presence of SLE virus in pools of homogenized mosquitoes. This approach could provide early data on which to base disease control decisions.