Among the EF-hand family type of calcium-binding proteins (CaBPs) calbindin-D28k and calretinin are abundant in the nervous system and show a specific distribution. They have been demonstrated in many regions of the brain and they often can be found in certain populations within a given nucleus. Although their specific functional role is not quite clear, they have been used in anatomical and developmental studies as markers for analyzing neuronal subpopulations. It is commonly accepted that CaBPs are important for buffering intracellular free calcium, and, in this respect, they are probably significantly important in some neuronal elements of the highly-active auditory system. The aim of our study was to determine the distribution of calbindin-D28k (CB) and calretinin (CR) in the auditory pathway of the posthatch chicken.Within the auditory nuclei of the brainstem clear differences between and within the nuclei were found. Whereas in the first nucleus of the intensity pathway, the nucleus angularis, a subpopulation of CR immunoreactive (CR-IR) neurons with a higher number in the dorsolateral part was found, most or all cells of the nuclei of the time pathway, nucleus magnocellularis and nucleus laminaris, showed CR-IR. In the superior olive several fibers were stained for CR-IR. Interestingly, in rare cases neurons surrounding the SO with dendritic processes within the SO were stained for CB immunoreactivity. Investigation of CR staining in the nucleus of the lateral lemniscus revealed a clear division in a dorsal and a ventral part, thus being a powerful tool for visualizing these subnuclei, which is difficult when using other staining methods. In the inferior colliculus (MLd) a complex pattern of distribution of CaBP-IR was found confirming the classification of this nucleus as suggested by Puelles et al. (JCN 340:98-125, 1994).In summary, the distribution of CaBPs in the auditory pathway of the chicken reveals distinct inter- and intranuclear differences providing us with a powerful tool for understanding the architectural and functional significance of neuronal subpopulations.Supported by a travel fellowship to AK by the Boehringer Ingelheim Fonds.