Rat alpha-fetoprotein (AFP) contains one binding site for estradiol with an association constant, Ka = 1 X 10(8) M-1. This site overlaps with a high affinity arachidonic acid binding site (Ka = 1 X 10(7) M-1) and with a tryptophan methyl ester binding site, AFP also possesses a second site able to bind retinoids with a Ka = 1 X 10(6) M-1, and a third site for the binding of bilirubin (Ka = 1 X 10(6) M-1). These two sites presented no interaction and do not overlap with the estrogen or fatty acid binding site. It is suggested that these three binding sites reflect the three-domain structure of the rat AFP molecule deduced from the studies on the nucleotide sequence of AFPmRNA and AFPcDNA segments.