Hyperbilirubinemic Gunn rats were used to study sulfisoxazole‐mediated displacement of bilirubin from albumin. Bilirubin not bound to albumin or unbound bilirubin, which can enter the brain and cause damage, was measured by the automated peroxidase micromethod using the UB‐Analyzer. When sulfisoxazole was added to the rat serum in vitro, it showed measurable displacing competition with bilirubin for the binding sites on albumin. The bilirubin titration curves of hyperbilirubinemic sera, after injection of sulfisoxazole, showed a shift to the left indicating that the drug effectively lowered the capacity of serum albumin to bind to bilirubin. The bilirubin binding affinity of the first binding site on the albumin decreased with the administration of 25 mg/kg or more of sulfixazole. The serum concentration of both total bilirubin and unbound bilirubin decreased after the injection of sulfisoxazole suggesting their diffusion into extravascular compartments. Copyright © 1983, Wiley Blackwell. All rights reserved