Mechanisms underlying bilirubin encephalopathy and hearing loss remain poorly understood, including the way bilirubin enters the nervous system and how bilirubin accumulates in circumscribed regions of the brain. The present experiments examined the auditory brainstem in heterozygous (Nj) and homozygous (jj) Gunn rats at an age when serum bilirubin levels were highest, and after brain bilirubin concentration was artificially raised by sulfadimethoxine administration. In four litters of 11-12 days old Gunn rats, Nj and jj littermates received a single intraperitoneal injection of sulfadimethoxine (100 mg/kg) or a comparable volume of saline. At 16-17 days of age, brainstem auditory evoked potentials were recorded to assess the severity of bilirubin toxicity in the Nj and jj animals. Following the recordings, each animal was perfusion-fixed and frozen sections of the brainstem were cut in the transverse plane from medullary through mesencephalic levels. Sections were mounted on slides, stained with thionin and coded to avoid observer bias. Quantitative analysis revealed no differences between saline and sulfa-treated Nj rats for cochlear nucleus volume, or for cell size in the cochlear nucleus or superior olive. In the sulfa-treated jj rats, cochlear nucleus volume, and cross-sectional areas of spherical cells in the anteroventral cochlear nucleus and principal cells in the nucleus of the trapezoid body, were all significantly smaller than in the combined groups of Nj animals. The affected areas in the cochlear nucleus and superior olive are innervated by large axosomatic end-bulbs of Held or calyceal endings, and were associated with bilirubin staining of glia in the most severely jaundiced jj sulfa-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)