Geckos use vocalizations for intraspecific communication, but little is known about the organization of their central auditory system. We therefore used antibodies against the calcium-binding proteins calretinin (CR), parvalbumin (PV), and calbindin-D28k (CB) to characterize the gecko auditory system. We also examined expression of both glutamic acid decarboxlase (GAD) and synaptic vesicle protein (SV2). Western blots showed that these antibodies are specific to gecko brain. All three calcium-binding proteins were expressed in the auditory nerve, and CR immunoreactivity labeled the first-order nuclei and delineated the terminal fields associated with the ascending projections from the first-order auditory nuclei. PV expression characterized the superior olivary nuclei, whereas GAD immunoreactivity characterized many neurons in the nucleus of the lateral lemniscus and some neurons in the torus semicircularis. In the auditory midbrain, the distribution of CR, PV, and CB characterized divisions within the central nucleus of the torus semicircularis. All three calcium-binding proteins were expressed in nucleus medialis of the thalamus. These expression patterns are similar to those described for other vertebrates. J. Comp. Neurol. 518:3409–3426, 2010. © 2010 Wiley-Liss, Inc.