Concurrent recordings of bat calls at the ground and canopy levels were compared to analyse the vertical stratification of habitat use in a broad-leaved deciduous forest in central Europe. The recording effort was 48 nights, by sampling 16 sites three times in 2010. Overall, 2170 call sequences were recorded; 40% of them at the canopy level. Sixteen bat species were identified, 13 of which occurred at ground level and 14 in the canopy. By fitting generalized linear mixed models for seven species and all calls sequences combined, the effects of stratum, time period, ambient temperature and forest structure on bat-calling activity were assessed. Four species preferred the ground level (Barbastella barbastellus,Eptesicus serotinus,Myotis daubentonii,Pipistrellus pygmaeus). M. bechsteinii was the only species which had a significant preference for the canopy level in the pregnancy, lactation and post-lactation period, whereas two further species (M. alcathoe,P. pipistrellus) showed a significant canopy preference in at least one period. Therefore, canopy sampling should be undertaken when aiming at monitoring such species for conservation purposes. Populations of certain target species will be underestimated by ground recordings alone.