The origins of steroid-dependent regulation of the vertebrate estrogen receptor (ER) are poorly understood. We used artificial-intelligence-based software to construct 12 motifs specific to the estrogen-binding domain of ER alpha and ER beta in land vertebrates and teleosts. We mapped these ER-specific motifs onto the sequences of lamprey, amphioxus, and invertebrate and selected vertebrate ERs and estrogen-related receptors (ERR). Lamprey ER contains 11 motifs common to ERs in the training set. In contrast, amphioxus ER contains only six motifs. Unexpectedly, human and amphioxus ERRs contain nine motifs. We mapped the 12 motifs onto an alignment of human, lamprey and amphioxus ERs, which depicted residues in human ER alpha that interact with estradiol, which revealed significant differences between amphioxus ER and vertebrate ERs in the steroid-binding domain. This suggests unusual ligand recognition in amphioxus ER. (c) 2008 Elsevier Inc. All rights reserved.