Sequences of the advertisement calls produced by male Nathusius' pipistrelles (Pipistrellus nathusii) during the autumn mating period were recorded from individuals at two separate sites in Antrim, Northern Ireland, in August 2004. Several male roosts were found at these sites in close proximity to a single maternity roost, each containing approximately 200 adult females and their young. Analysis of measured parameters of four identified call types revealed that there were significant differences in call structure between sites and between individuals. Playback experiments, performed outside the adult female and juvenile roost sites, comprised of experimental advertisement call sequences of P. nathusii, Pipistrellus pygmaeus and Pipistrellus pipistrellus and control sound recorded without bats present (silence). Response was measured by simultaneously recording ultrasound during playbacks and counting the number of echolocation pulses identified as those of P. nathusii above a predetermined amplitude threshold. Significantly greater numbers of P. nathusii echolocation pulses were recorded during playback of male P. nathusii advertisement calls than during playback of congeners' advertisement calls and control sound. The number of echolocation pulses recorded was similar during playback of P. pipistrellus and P. pygmaeus advertisement calls and silence. We suggest that, due to call complexity, male P. nathusii advertisement calls should be classified as ‘song'. Species-specificity and individual variation suggests that the songs of male P. nathusii have the potential to play a role in mate attraction and mate assessment.