alpha-Fetoproteins (AFP) produced in rodents bind estrogen, while those in other species including human do not. The estrogen binding region of rat AFP has been identified to locate at a segment of amino acid residue 426-467 in the third domain from the experiment using yeast recombinant rat-human chimeric AFPs from this laboratory. In the region, 15 amino acid substitutions were observed between rat and human AFP, and, therefore, the residues responsible for estrogen binding should be contained among them. In order to identify the amino acid residues needed for estrogen binding, a series of mutant human AFPs in which some human-specific amino acid residues were substituted with those of rat AFP were produced by site-directed mutagenesis employing polymerase chain reaction. Among 17 mutant human AFPs carrying various combinations of substitutions, those carrying substitutions 436Ala-->Val, 437Ala-->Ser, 438Thr-->Ile, 450Leu-->Arg, 451Leu-->Ser, 457Ala-->Leu, and 461Ile-->Tyr showed significant estrogen binding activities. Human AFP, originally unable to bind estrogen, could be converted to a variant which binds estrogen by introducing these substitutions. The mutant human AFP could be used to modulate the biological activity of estrogen as reported for rat AFP.