The rabbit alphas1-casein gene contains a distal prolactin-dependent enhancer 3442-3285 bp 5' to the site of initiation of transcription. We have reported previously that four DNA/protein-binding sites (F1-F4) are located within this distal enhancer. We now show that one of this binding site (the F4 site) binds in vitro a MGF/STAT5-like factor. The functional importance of the F4 site was estimated by cotransfection of CHO cells with a chimeric gene containing or not the F4 sequence linked to the (-391/+1774)CAT gene and a plasmid encoding the rabbit mammary prolactin receptor. The F4 site is necessary for maximal response, of the enhancer to prolactin. However, this site has to be associated to the Fl-F3 fragment. It can be replaced by a genuine MGF/STAT5-binding site. A mutational analysis indicates that F4 and F1 sites are simultaneously involved to confer a high prolactin sensitivity.