The estrogen receptor (ER) binds with high affinity to the nonclassical estrogen response elements (ERE) found in the rat prolactin gene. There are two putative EREs in this gene; at -1582 and -1722 upstream of the transcriptional start site. We used DNA binding assays based on the immunoprecipitation of receptor with bound DNA to quantitate the binding of ER to these two elements. ER bound each element with significantly higher affinity than it bound to nonspecific DNA, but with 10-100-fold less affinity than that for the classical ERE sequence derived from the vitellogenin A2 gene. These comparisons rank the prolactin gene sequences as weak EREs. We also observed a 1:1 ratio of ER to ERE in the bound complexes, indicating that these high-affinity interactions were not dependent upon an ER homodimer. These data support the role of these sequences in mediating estrogen regulation of the prolactin gene.