Fixed site attachment and a long calling period within an environment of little temperature change render the males of the diurnal Pan-Amazonian frog Allobates femoralis a rewarding species for field studies on both calling and hearing in anurans. The terrestrial calling positions are spaced at several meters, thus allowing to move within the territories without disturbing the resident males. Vocally active males react with stereotypic phonotactic responses to the playback of conspecific and a large variety of synthetic calls. To test for interference effects of co-occurring species on the male-male communication system of A. femoralis, we compared the calls and the phonotactic reaction of territorial males at eight Amazonian sites: four where A. femoralis occurs together with Ameerega trivittata and four where A. trivittata is absent. The occurrence of A. trivittata apparently gave rise to narrower and asymmetric frequency-response curves in A. femoralis, without concomitant differences in the call or in body size. Playback experiments have also allowed to disentangle the relative contribution of visual and auditory cues in multimodal signalling. To investigate the relative contributions of different evolutionary mechanisms to geographic differentiation of these populations, we compared calls, colouration, body shape, and the cyt-b gene. The studied traits did not evolve as a unit. Against our expectations and in contrast to current models of speciation in the Amazon basin, a significant amount of geographic variation in calls and cyt-b was attributable to geographic distance between populations. To the best of our knowledge, up to now A. femoralis has been among the most intensively studied anuran species under completely natural conditions, i.e. without touching the frogs prior to and during the acoustic field experiments.