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We report the first seroprevalence study of the occurrence of specific antibodies to European bat lyssavirus type 2 (EBLV-2) in Daubenton bats. Bats were captured from 19 sites across eastern and southern Scotland. Samples from 198 Daubenton bats, 20 Natterer bats, and 6 Pipistrelle bats were investigated for the presence of EBLV-2. Blood samples (N = 94) were subjected to a modified fluorescent antibody virus neutralization test to determine antibody titer. From 0.05% to 3.8% (95% confidence interval) of Daubenton bats were seropositive. Antibodies to EBLV-2 were not detected in the 2 other species tested. Mouth swabs (N = 218) were obtained, and RNA was extracted for a reverse transcription–polymerase chain reaction (RT-PCR). The RT-PCR included pan lyssavirus-primers (N gene) and internal PCR control primers for ribosomal RNA. EBLV-2 RNA was not detected in any of the saliva samples tested, and live virus was not detected in virus isolation tests.