A diazo-positive fraction of serum bilirubin that is irreversibly bound to albumin has been shown to accumulate in serum of patients with cholestasis. In the present study, a cholestatic animal model was used to determine the chemical nature of the bilirubin species involved in its formation. The data indicate that conjugated bilirubin is the precursor of "albumin-bound bilirubin" and that the presence or absence of light does not affect its formation. An albumin-bound bilirubin-complex indistinguishable from the complex detected in cholestatic sera from patients or in bile duct-ligated Sprague-Dawley rats can be formed in vitro in sera enriched in conjugated bilirubin at 37 degrees C, pH 7.4.