Previous studies have shown that estrogen responsiveness of the rat PRL gene requires the presence of both the estrogen receptor and the tissue-specific transcription factor, Pit-1. To examine the contribution of individual Pit-1-binding sites in permitting an estrogen response, we mutated specific sites in both the proximal and distal regions of the rat PRL gene. The studies reveal that mutation of Pit-1-binding sites in either the proximal or the distal region can have an effect on estrogen responsiveness. The most important Pit-1-binding site appears to be the site in the distal enhancer, which is adjacent to the estrogen receptor-binding site. However, mutation of combinations of other Pit-1-binding sites reveals that these sites also contribute to the estrogen response of the PRL gene. The binding sequences for another transcription factor cannot substitute for Pit-1 sites in bringing about a wild-type estrogen response, as shown by replacement of Pit-1-binding sites with a consensus cAMP-responsive element. Conversion of the imperfect palindromic estrogen response element of the PRL gene to a perfect palindrome eliminated the positive effects of an intact 1D Pit-1-binding site. To examine potential physical interactions between the estrogen receptor and Pit-1, a protein interaction assay was performed. The results demonstrate that labeled estrogen receptor can bind to Pit-1 immobilized on glutathione agarose beads. However, most of the interaction between Pit-1 and the estrogen receptor appears to be DNA dependent.(ABSTRACT TRUNCATED AT 250 WORDS)