To evaluate the relationship of serum cerebrospinal fluid (CSF) and brain total bilirubin levels in asphyxia, an experiment was designed with 5 to 6-week-old Sprague-Dawley rats. The rats were randomized into control and experimental groups. All rats received intravenously 30 mg/kg of bilirubin. Four hours later the experimental group was asphyxiated. Forty-eight hours after asphyxiation, the bilirubin concentrations in blood, CSF, and brain were measured in both study groups. Mean CSF and brain bilirubin levels were significantly higher in the experimental compared to the control group; however, mean serum bilirubin levels were not different. Moreover, in the experimental group a significant correlation existed between CSF and brain bilirubin concentrations. In conclusion, an asphyxiatic insult resulted in disruption of both the blood-brain and the blood-CSF barriers.