The dorsal nucleus of the lateral lemniscus (DNLL) is an auditory structure of the brainstem. It plays an important role in binaural processing and sound localization and it provides the inferior colliculus with an inhibitory projection. The DNLL is a highly conserved auditory structure across mammals, but differences among species in its detailed organization have been reported. The main goal of this study was to analyze the topographic organization of the cat DNLL. Single, small iontophoretic injections of biotinylated dextran amine were made at different loci in the central nucleus of the inferior colliculus (CNIC). The distribution of the labeled structures in the ipsi- and contralateral DNLL was computer reconstructed in three dimensions. In individual sections, a band of labeling is seen in the DNLL on both sides. These two labeled bands occupy symmetric locations and are made of retrogradely labeled neurons with flattened dendritic arbors oriented parallel to each other. Moreover, the ipsilateral labeled band contains labeled terminal fibers parallel to the labeled dendrites. With three-dimensional reconstructions, it becomes evident that the labeled band seen in each individual DNLL section represents a slice through a rostrocaudally oriented lamina. The shape, size, orientation, and location of this lamina change as the injection site is shifted along the tonotopic axis of the CNIC. An injection in the low-frequency region of the CNIC, produces a lamina that resembles a flattened tube located in the dorsolateral corner of the DNLL. An injection in the high-frequency region of the CNIC, by contrast, results in a lamina that is an elongated sheet located at the ventromedial surface of the DNLL. The laminae of the DNLL might constitute the structural basis for its tonotopical organization. Previous studies (Merchan MA, et al. 1994. J Comp Neurol 342:259-278) in conjunction with our current results suggest that the laminar organization in the DNLL might be common among mammals.