The central nucleus of the inferior colliculus (CNIC) is comprised of an orderly series of fibrodendritic layers. These layers include integrative circuitry for as many as 13 different ascending auditory pathways, each tonotopically ordered. Calcium-binding proteins, such as calbindin-D28k (CB), may be useful neurochemical markers for specific subsets of afferent input in these layers and their spatial organization that are developmentally regulated. In this study, CB-immunohistochemistry was used to examine 1–42 postnatal-day-old kitten and adult cat CNIC and anterograde tracers were used to label afferent projections from the lateral superior olivary nucleus (LSO) to the CNIC at similar ages. A distinct axonal plexus that is CB-immunopositive is described. This CB-afferent compartment is present at birth and persists throughout the ages examined. Already at birth, the CB-immunostained plexus in kitten CNIC is organized into discrete bands that are approximately 75 μm thick and 500 μm long. In adult CNIC, the periodic banded pattern of CB-immunostained fibers is similar to that in kittens albeit bands are thicker (145 μm) and longer (700 μm). Growth in band thickness in adult cat appears proportional to growth of the IC, whereas length of the dense CB-immunostained bands is somewhat more focused in the central region of fibrodendritic layers. The banded pattern of the CB-immunostained plexus is well correlated with the location and dimension of afferent projections from the LSO in newborn kitten labeled with carbocyanine dye, 1,1′-dioctodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate and in adult cat labeled with wheat germ agglutinin conjugated with horseradish peroxidase. The results reveal a neurochemical marker for one type of synaptic compartment in CNIC layers, banding, that is organized before hearing onset in kittens, but that may undergo some postnatal pruning.