Sensorineural hearing loss and auditory dysfunction are major sequelae of neonatal hyperbilirubinemia. The sites and cellular effects of bilirubin toxicity in the auditory brainstem pathway are not easily detected. Since altered intracellular calcium homeostasis may play a role in neuronal cell death, we hypothesized that the expression of calcium-binding proteins may be altered in the classic animal model of bilirubin neurotoxicity. The expression of the calcium-binding proteins, calbindin-D28k and parvalbumin, in the brainstem auditory pathway of homozygous recessive jaundiced (jj) Gunn rats was examined by light and electron microscopic immunohistochemistry at 18 days postnatally and compared to the findings obtained from age-matched non-jaundiced heterozygous (Nj) littermate control rats. Immunoreactive staining for both calbindin and parvalbumin was reduced in the cochlear nuclei and the superior olivary complex in jj rats. The extent of the reduction in immunoreactivity was related to the severity of the clinical symptoms. By contrast, immunoreactive staining in other brainstem areas (e.g., dorsal and ventral nuclei of the lateral lemniscus, inferior colliculus), thalamic (medial geniculate body) auditory areas, and neighboring non-auditory structures was similar in jaundiced and control rats. Calbindin-immunoreactive staining in the superior paraolivary and medial superior olivary nuclei in Nj rats was associated with myelinated axons, whereas parvalbumin-immunoreactive staining was localized postsynaptically in neuronal somata and dendrites. Immunoreactive staining for the calcium-binding proteins calbindin and parvalbumin in lower brainstem auditory nuclei shows abnormalities in areas susceptible to the effects of hyperbilirubinemia and provides a sensitive new way to assess bilirubin toxicity in the auditory system.