Ascending projections of the cochlear nuclei in the mustache bat were analyzed by anterograde transport of [3H]-leucine and by retrograde transport of HRP. We were particularly interested in pathways to two parts of the system: (1) to the medial superior olive, because this nucleus is missing in most echolocating bats, but appears to be present in the mustache bat, and (2) to the intermediate and ventral nuclei of the lateral lemniscus, because these nuclei are hypertrophied and highly differentiated in all echolocating bats that we have examined. The results show a highly systematic projection from the anteroventral cochlear nucleus to all of the auditory nuclei in the brain stem. After an injection of [3H]-leucine in the anterior and dorsal part of the anteroventral cochlear nucleus, presumably in a region sensitive to low frequencies, label is seen in the following locations: ipsilateral to the injection in the lateral part of the lateral superior olive; bilaterally in the dorsal part of the medial superior olive; contralateral to the injection in the dorsal parts of the intermediate and ventral nuclei of the lateral lemniscus; and in the anterolateral part of the central nucleus of the inferior colliculus. After an injection of [3H]-leucine in a posterior part of the anteroventral cochlear nucleus, presumably in a region sensitive to high frequencies, labeling is in the same set of nuclei, but within each nucleus the label is now in a different location: medially in the lateral superior olive, ventrally in the medial superior olive, ventrally in each division of the ventral and intermediate nuclei of the lateral lemniscus, and medially in the central nucleus of the inferior colliculus. Projections from the entire anteroventral cochlear nucleus to the inferior colliculus are confined to the ventral two-thirds of the central nucleus. The dorsal one-third of the central nucleus of the inferior colliculus is the principal target of the dorsal cochlear nucleus and may be a target of the posteroventral cochlear nucleus. Both of these nuclei appear to project sparsely to the ventral parts of the inferior colliculus. We conclude first that the bilateral input to the medial superior olive in the mustache bat is similar to the input seen in other mammals. Thus this bat has a neural structure which is associated with the analysis of binaural time differences and which usually is seen only in animals with heads large enough to create interaural time differences greater than those available to Pteronotus.(ABSTRACT TRUNCATED AT 400 WORDS)