The serotine bat (Eptesicus serotinus) accounts for 95 % of cases of human exposition to EBLV1. The aim of this study was to focus on the epidemiology and pathogenesis of EBLV1 infection in the serotine bat. Our first objective was the development of an RT-PCR technique for the specific detection of EBLV1 RNA in oro-pharyngeal swabs. This technique showed better performance than the classical immunofluorescence (IF) on brain in detecting EBLV1 in healthy flying bats. We have used this technique together with antibody detection by the fluorescent focus inhibition test (FFIT) to investigate EBLV1 circulation in 19 natural colonies of serotine bats in Andalusia (Spain) from 1998 to 2003. The survey was based on 1223 different captures of 1080 individuals that were ring banded, sampled and released. Individuals that were repeatedly captured were always found in the same colony even though some colonies were less than five Km apart. Viral circulation was detected in nine colonies either by RT-PCR, serology or both. Each colony showed a different temporal pattern of viral circulation suggesting independent endemic circulation. Some positive individuals were captured healthy in following campaigns evidencing survival after viral infection.