P-glycoprotein, the product of the multidrug resistance (mdr) gene family, is a major determinant in the development of resistance to a large number of cancer chemotherapeutic agents and is also expressed normally in a variety of mammalian tissues. In rodents during pregnancy, there is a dramatic overproduction of the mdr1b form of P-glycoprotein at the lumenal surface of the secretory epithelium of the gravid uterus. An expression vector, mdr1b-CAT, was constructed by fusion of this promoter region to a reporter gene, the bacterial chloramphenicol acetyltransferase (CAT) gene. R5020, a progesterone agonist, increased approximately 3-fold the expression of mdr1b-CAT when transfected into T47D cells, a cell line that constitutively expresses the progesterone receptor. A far greater response to R5020 was observed when the cells were co-transfected with an expression vector for the A form of the progesterone receptor, but not the B form. A series of 5'-deleted clones of the mdr1b-CAT construct indicated that the region of responsiveness was located in the first untranslated exon of the gene. Furthermore, sequences from the first exon were able to confer responsiveness to the non-responsive thymidine kinase-CAT vector. This study demonstrates that progesterone specifically regulates the activity of the mdr1b promoter and that this response is directed solely by the A form of the progesterone receptor.