Cicada barbara lusitanica males presented a stereotyped singing response behaviour when exposed to a playback of the conspecific song. Males preferred (as measured by the time taken to sing) the conspecific signal to heterospecific songs that differed markedly in temporal pattern. Manipulation of the gross temporal pattern of C. barbara calling song significantly reduced stimulus attractiveness. Indeed, C. barbara males stopped responding to stimuli in which the temporal pattern approached the characteristic C. orni song, a sympatric and closely related species. If present in females, the preference for stimuli with pauses not exceeding 30 ms could reflect the evolution of a behavioural pre-copulatory isolating mechanism based on song analysis. Males discriminated frequencies within 3-15 kHz, clearly preferring 6 and 9 kHz; both these frequencies matched the main spectral peaks of the song. The preference for specific frequencies was not associated with maximum neuronal excitation as estimated by auditory nerve recordings, which suggests that this frequency-dependent behaviour is not based on the strength of the auditory system's response to different frequencies. Rather, it is likely to reflect fine frequency resolution in the central nervous system.