To determine the prevalence of European Bat Lyssavirus 1 (EBL1), antibodies plasma samples were obtained from 175 serotine bats (Eptesicus serotinus) from four colonies in southern Spain between September of 1991 and September 1992. Five bats were detected with EBL1 virus in one colony in 1989. The prevalence of antibodies rose to 74% in one of the colonies studied (Villarrasa) in the spring of 1992. After a few months the prevalence declined to under 10%. Individuals with a high antibody level in the spring (up to ED50 = 280) had very low titers or no antibodies in the following summer and autumn.