The inferior colliculus of the mustache bat is similar in most respects to the inferior colliculus of more commonly studied mammals, but one isofrequency contour, the dorsoposterior division, is greatly overrepresented. The present study utilizes this specialization of the auditory system in the mustache bat to determine the total set of ascending projections to a single isofrequency contour of the inferior colliculus. Within the dorsoposterior division, neurons are all very narrowly tuned to 60 kHz, the major component of this bat's echolocation call. The afferent projections to this isofrequency contour were identified by making deposits of horseradish peroxidase (HRP) within the dorsoposterior division after physiologically defining its borders. Two other frequency representations are present in the central nucleus of the inferior colliculus of the mustache bat, the anterolateral division in which there is an orderly progression of frequencies from 59 down to 20 kHz, and the medial division in which frequencies from 63-120 kHz are represented. In additional experiments, the afferent projections to the medial and anterolateral divisions were examined, providing an anatomical description of the tonotopicity of the lower auditory nuclei. Deposits of HRP in the DPD labeled cells in each of the lower brainstem auditory nuclei that have previously been shown to project to the entire central nucleus of the inferior colliculus. The ascending projections to the dorsoposterior division include contralateral projections from the cochlear nucleus and inferior colliculus, ipsilateral projections from the medial superior olive, ventral and intermediate nuclei of the lateral lemniscus, and bilateral projections from the lateral superior olive and dorsal nucleus of the lateral lemniscus. In most of the nuclei, labeled cells were confined to specific portions of the nuclei, often forming "slabs" of labeled cells across the rostrocaudal extent of most nuclei. These slabs presumably represent the 60 kHz representation in each of the lower nuclei. When deposits of HRP were made into other frequency band representations of the inferior colliculus, in either the medial or anterolateral division, labeled cells again formed slabs in each lower nucleus. However, the location of the slab varied as a function of the best frequency of neurons at the deposit site, and labeled cells were not present within the 60 kHz representation. These results show the general tonotopy of the mustache bat's brainstem auditory nuclei, and with respect to the dorsoposterior division, clearly reveal the total set of projections to a single isofrequency region.(ABSTRACT TRUNCATED AT 400 WORDS)