The ammalian ear contains two types of auditory receptors, inner and outer hair cells, that lie in dose proximity to each other within the sensory epithelium of the cochlea. In adult mammals, these two classes of auditory hair cells are innervated by separate populations ofafferent neurons that differ strikingly in their cellular morphology and their pattern of arborization within the cochlea. At present, it Is unclear when or how these disinctive patterns of cochlear innervation emerge and become segregated during development. In the present study, an in vitro horseradish peroxidase labeling method was used to examine the formation of individual auditory neuron arbors at the same location within the apex of the developing gerbil cochlea. At birth, most cochlear neurons displayed peripheral arbors that embraced both inner and outer hair cell receptors. During the next 6 days, however, the arbors of individual cochlear afferents become confined to either the inner or outer hair cell zone, and thus there is a complete segregation of afferent innervation. This neural segregation occurs principally through the withdrawal of inappropriate connectimns to the outer hair cell ystem and is completed well before hearing commences.A central goal of neurobiology is to determine how developing neurons choose their specific synaptic partners. An excellent system for examining this issue is the mammalian auditory endorgan, the cochlea, since its pattern of innervation is among the most precise of any sensory or motor system. The adult cochlea contains two types of auditory receptors, termed inner (IHCs) and outer (OHCs) hair cells, which receive separate and highly distinctive patterns of afferent innervation. Ninety percent of all cochlear afferents project to individual IHCs via thick unbranched radial afferents, each of which terminates in a single synapse. The OHCs, on the other hand, receive a more diffuse innervation from a relatively few thin spiral afferents, each of which projects for hundreds of micrometers along the length of cochlea before branching to contact from 5 to 50 receptorsUltrastructural studies have provided evidence for substantial ontogenic changes in the form, number, and spatial arrangement of synapses between mammalian auditory receptors and cochlear nerve fibers (9-16). To understand the process by which these synaptic patterns are adjusted during development, however, it is necessary to examine the early morphological changes that occur in the formation of indi- In the present investigation, I have employed an in vitro horseradish peroxidase (HRP) method that permits labeling of small populations of auditory nerve fibers at any location within the developing cochlea. The Mongolian gerbil was selected for study since this species displays a prolonged period of postnatal cochlear maturation. In addition, the gerbil cochlea, like that of the mouse (26), lengthens <10% after birth, from an average of 10.8 mm in the newborn (unpublished data) to 12.1 mm in the adult (27), which greatly reduces ...