Vocalizations of Boophis madagascariensis (Rhacophoridae) males were recorded in a mid-elevation rain forest in eastern Madagascar. Call notes made by males of this species were classified into 28 types. This represents the largest known call repertoire of any amphibian. The calls range widely in spectral characteristics from a narrowband, nearly pure-tone to broadband ‘rip’ notes, to even broader band, pulsatile ‘iambic’ notes consisting of up to 23 pulses. Even considering all ‘iambic’ notes as variants of one type, there remain eight distinct call notes produced by this species. Morphological analysis of the vocal musculature of B. madagascariensis revealed no obvious adaptations for unusual glottal flexibility or function. Playback studies using a subset of the call notes as stimuli were carried out in the animals' natural habitat to determine the functional significance of several of the call notes. At the lowest playback levels, there was a significant tendency for males to produce rip notes in immediate response to either rip or iambic note stimuli. At the higher playback levels, we identified one sequence of iambic notes that consistently evoked a significant iambic note response from males. A permutation test on call note transitions during spontaneous calling revealed that call note pairs occurred inconsistently and unpredictably. The evolutionary significance of an extended vocal repertoire, matched counter-calling and a random call note production scheme is discussed.