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In this study, we analysed the patterns of variation of the European treefrog’s advertisement call at four levels of organization: within individuals, within populations, among populations of the same species, and among different species of the same clade. At the within-individual level, call acoustic properties are distinguished into static and dynamic properties. At the within-population level, two sources of call variation were analysed: temperature and body size. Temperature affects both temporal and spectral properties of the call. Body size mostly affects spectral properties. At the between-population level, calls do not show significant differences with respect to temporal properties, but they do differ in two spectral (and stereotyped) properties: the fundamental frequency and the difference in amplitude between dominant and fundamental frequencies. Finally, at the between-species level, call differences are much more conspicuous: they involve both spectral and temporal and both static and dynamic properties. At all four levels, body size is associated with call variation, explaining 11% of the differences among populations of the same species and 73% of the differences among species of the same clade. On the basis of these results, we hypothesize that patterns of variation of call acoustic properties, their constraints, and their biological functions are intrinsically associated. We discuss the role that such an association might play in the evolution of acoustic signals.