The mitogen-regulated protein/proliferin (mrp/ plf) genes belong to the prolactin/growth hormone gene superfamily and encode at least four closely related proteins. Identified functions of these proteins include stimulation of uterine proliferation and endothelial angiogenesis. In 3T3 cells, basic fibroblast growth factor (bFGF) stimulates the production of mrp/plf mRNAs with a resulting increase in the protein products. Although the three cloned mrp/ plf gene promoters (mrp3, plf42, and plf149 ) are over 97% identical in sequence, only mrp3 is transcriptionally activated by bFGF. We have identified a sequence in the mrp3 promoter, which we have named the "bFGF-responsive element" (FRE), (-186 to -167), that specifically binds nuclear factors from 3T3, CHO and Hela cells. Analysis of the bFGF-responsiveness of a series of truncated mrp3 promoter sequences combined with footprint analysis, pinpointed a region of the promoter that contains a large variation in sequence between the three promoters and one base in the sequence that is unique to the mrp3. The nuclear factors bound by the FRE are present in the placenta and the fetus in which the gene is expressed. By contrast, the maternal liver does not contain FRE-binding proteins. The FRE is transcriptionally active in a TK fusion promoter and responds to bFGF in this context. Our results show that the FRE is an bFGF-responsive transcriptional element. We demonstrate the sequence between -186 to -167 of the three mrp/plf promoters is differentially regulated by bFGF in a TK fusion promoter. Characterization of the 31--36 kDa protein(s) which bind these sequences suggest that different high affinity binding proteins bind to these different sequences. The core sequence of the FRE is also found in the promoters of genes encoding the interstitial collagenase type I and stromelysin-1, that are also regulated by bFGF as delayed early response genes. The FRE element may be the means by which the expression of mrp3 and other genes are regulated by bFGF