Flanders virus was isolated from multiple pools of Culex pipiens complex and Culex restuans mosquitoes collected during 1965–1967 at several localities in the Ohio-Mississippi Basin. In this region, the virus was detected earlier in the mosquito-breeding season than was St. Louis encephalitis virus. Moreover, the presence of Flanders virus was not related to the hot and dry weather that appeared to favor SLE virus. As a result of studies in McLeansboro, Illinois, 14 strains of Flanders virus were isolated from blood taken from birds collected in 1967 during the period when isolation rates were highest in mosquito pools. Mosquito-bird-mosquito transmission cycles were active, but there was no evidence that human residents were infected.