We have previously shown that a human mammotropic polypeptide hormone, prolactin (PRL) can act synergistically with steroid hormones to regulate gene expression directed by the long terminal repeat of mouse mammary tumor virus (MMTV LTR) in a human ductal carcinoma cell line T47D cells using a chloramphenicol acetyltransferase reporter gene system and gene transfection methods. In the present study, using various recombinant plasmids we analyzed functional elements in the MMTV LTR that is essential for the PRL responses. We show that the PRL-responsive elements are located in the extreme 5' end of the MMTV LTR, a region previously described by others to be a mammary cell-specific enhancer.