Inoculation of the big brown bat (Eptesicus fuscus) with a small dose of a St. Louis encephalitis (SLE) virus strain isolated in Ohio indicated that the big brown bat was susceptible to infection. The virus was maintained in the bats through hibernation (70 days), and the bats developed a viremia within 4 days of arousal from hibernation (105 days after inoculation). A field survey of 390 big brown bats and little brown bats (Myotis lucifugus) conducted in 5 regions of Ohio during 1979 to 1981 revealed a SLE virus-neutralizing antibody prevalence of 9%. Cohabitation of natural caves and abandoned mineshafts in Ohio by Culex pipiens mosquitoes, big brown bats, and little brown bats was also documented. Demonstration of a 9% prevalence rate of neutralizing antibody to SLE virus in big brown bats and little brown bats in Ohio during a nonepizootic period indicated that the bat may be involved in the maintenance of SLE virus in enzootic foci and could have a role in dissemination of SLE virus to epizootic foci.