A powerful and versatile system for the identification of novel response elements for members of the intracellular receptor family is presented as applied to the human estrogen receptor. In the past, a limited number of estrogen response elements (EREs) have been functionally identified in the promoter regions of estrogen-regulated genes. From these a consensus ERE has been defined that is identical to the ERE of the Xenopus laevis vitellogenin gene, i.e., 5'-GGTCA NNN TGACC-3'. In order to investigate without bias the range of sequences that could function as EREs in vivo, we have developed a genetic selection in yeast expressing the human estrogen receptor (hER) and transformed with a random oligonucleotide library in a vector where expression of a selectable marker requires insertion of an upstream activating sequence. More than 1,000,000 transformants were screened and of 726 clones that contained activating sequences, 65 were found to be hormone-dependent. Sequencing revealed that the majority contained at least one 4/5 match to a canonical ERE half-site, but only one contained a full consensus ERE as previously defined. Some contained half-sites arranged as direct repeats. Twelve elements were further characterized to compare estrogen activation in yeast and mammalian cells and in vitro binding to hER. The results of these studies reveal that sequences that bind weakly to hER in vitro are fully functional as EREs in yeast and are conditionally responsive to estrogen in mammalian cells. In addition, an element was identified that is more sensitive to the partial agonist activities of tamoxifen and nafoxidine than is the consensus ERE, indicating that not only promoter context but the sequence of the binding site itself can allow distinction between receptor activated by agonist and that activated by antagonist.