The number of binding sites and the dissociation constants were determined for the binding of bilirubin to human liver ligandin and to human serum albumin. Albumin has a primary bilirubin binding site (KD = 0.03 microM), measured by the peroxidase procedure, and two apparently equivalent secondary binding sites (KD = 2 microM), determined by fluorescence quenching experiments. By contrast, ligandin does not have a corresponding high affinity site. The absence of this high affinity site was shown both by the peroxidase procedure and by direct competition between albumin and ligandin for bilirubin. Bilirubin binding to ligandin, measured by fluorescence quenching, is complex. At both pH 6.5 and 7.4, two interacting sites were observed with a Hill coefficient of 1.5, K' approximately 5 microM. Bilirubin binding to ligandin is not independent of glutathione S-transferase activity. Depending upon pH and upon the order with which the reactants are added, bilirubin can markedly alter the transferase activity. The results are interpreted in terms of kinetically stable conformational isomers of ligandin induced by bilirubin or by glutathione.