Severe hyperbilirubinemia in neonates with prematurity and/or systemic illnesses such as hemolytic disease, acidosis, and hypoxemia enhances their risk for developing cerebral palsy, paralysis of ocular upgaze, and deafness. This neurologic syndrome has been associated with selective neuronal vulnerability in the basal ganglia, certain brainstem nuclei, and Purkinje cells. However, the mechanism by which bilirubin damages neurons remains unclear. In these studies, we found that intracerebral injection of N-methyl-D-aspartate (NMDA), an excitotoxic analogue of glutamate, caused greater injury in jaundiced 7-day-old Gunn (jj) rat pups than in nonjaundiced heterozygous (Nj) littermate controls. NMDA injection caused even greater injury when protein-bound bilirubin was displaced with the sulfonamide drug sulfadimethoxine in jaundiced homozygous pups. In additional experiments, the acute signs of bilirubin-mediated neuronal injury, induced in homozygous (jj) Gunn rats by treatment with sulfonamide, were reduced by concurrent treatment with the NMDA-type glutamate channel antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine (MK-801, dizocilpine). The results suggest that bilirubin may cause encephalopathy and neuronal injury, at least in part, through an NMDA receptor-mediated excitotoxic mechanism. This conclusion is consistent with clinical observations that bilirubin encephalopathy is synergistically worsened by hypoxemia, which also shares an excitotoxic mechanism of neuronal injury.