The bilirubin-binding ability of human alpha-fetoproteins, which were purified from fetal cord serum and from ascites fluid of a hepatoma-bearing patient, was examined by the difference spectrum and the Jacobsen peroxidase methods. The difference spectrum observed as a result of the specific binding of bilirubin to alpha-fetoprotein had a maximum at 482 nm, and this pattern was quite similar to that observed for serum albumin. The result obtained by the difference spectrum method showed that 1 mol of each alpha-fetoprotein bound 1 mol of bilirubin at pH 8.3 and that the dissociation constants of the complexes of bilirubin with fetal alpha-fetoprotein and hepatoma-derived alpha-fetoprotein were 2.6 x 10(-7) and 5.0 x 10(-7) M, respectively. The Jacobsen enzymatic method using horseradish peroxidase gave the same values for molar binding ratios and similar dissociation constants, 7.1 x 10(-7) M for fetal alpha-fetoprotein and 7.4 x 10(-7) M for hepatoma-derived alpha-fetoprotein. These results indicate that alpha-fetoprotein may function as a carrier protein for bilirubin as has been shown for serum albumin.