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Mice produce various sounds within the ultrasonic range in social contexts. Although these sounds are often used as an index of sociability in biomedical research, their biological significance remains poorly understood. We previously showed that mice repeatedly produced calls in a sequence (i.e. calling bout), which can vary in their structure, such as Simple, Complex or Harmonics. In this study, we investigated the use of the three types of calling bouts in different sociosexual interactions, including both same- and opposite-sex contexts. In same-sex contexts, males typically produced a Simple calling bout, whereas females mostly produced a Complex one. By contrast, in the opposite-sex context, they produced all the three types of calling bouts, but the use of each calling type varied according to the progress and mode of sociosexual interaction (e.g. Harmonic calling bout was specifically produced during reproductive behaviour). These results indicate that mice change the structure of calling bout according to sociosexual contexts, suggesting the presence of multiple functional signals in their ultrasonic communication.