Parvalbumin (PV), calretinin (CR), and calbindin (CB) are calcium-binding proteins which are presumably involved in the regulation of the intracellular calcium concentration. Within the rat auditory system, CB is transiently expressed in several nuclei during the period of synapse refinement, indicating a specific function of CB during development, yet little is known in this regard about PV and CR. In order to gather more information about calcium-binding proteins during development, we analyzed the spatiotemporal distribution of PV and CR in the rat auditory brainstem using immunocytochemistry. In the adult, PV was heavily present in somata and neuropil of all nuclei and in fibers of all tracts. CR was found in somata of the cochlear nucleus and peripheral aspects of the inferior colliculus as well as in fibers extending into the superior olivary complex and the nuclei of the lateral lemniscus. The developmental expression of PV was characterized by a relatively late appearance in somata (at postnatal day 8), followed by a rapid increase to adult levels. In contrast, CR immunoreactivity was already strong two days before birth, yet the number and intensity of labeled neurons subsequently decreased and CR disappeared almost completely in the superior olivary complex, nuclei of the lateral lemniscus, and central aspects of the inferior colliculus. These data, together with those on CB, show that CR, CB, and PV are sequentially expressed during auditory brainstem development. They also suggest that the presence of the three proteins can be correlated with definite developmental stages.