Essential differences are demonstrated between bilirubin binding to rat serum proteins and to albumin in human serum. Acidimetric titration of rat serum with and without added bilirubin shows that binding of bilirubin acid in the range of pH from 6.8 to 8.8 takes place with release of less than one hydrogen ion per molecule of bound bilirubin. With human serum, two hydrogen ions are released, indicating binding of bilirubin dianion. The binding equilibrium of N-[4-[(4-aminophenyl)-sulfonyl]phenyl]-acetamide (MADDS) to rat serum albumin is influenced slightly by cobinding of bilirubin whereas MADDS and bilirubin bind competitively to human serum albumin. Finally, the rate of oxidation of bilirubin with hydrogen peroxide and peroxidase is decreased moderately by addition of rat serum albumin and strongly by the human protein, indicating that biliribin in its complex with rat serum albumin is subject to oxidation while the complex with human serum albumin is protected. These differences should be considered when rats are used as a model in experimental studies aiming at prevention of bilirubin encephalopathy in human neonates.