To examine some of the molecular mechanisms controlling transcription of the rat progesterone receptor (PR) gene, we have cloned and sequenced the 5'-region of the gene. Northern blot analyses with a series of probes identified two regions where distinct subsets of the multiple PR gene transcripts initiated, suggesting the presence of two promoters in the gene. Promoter activities for two gene fragments encompassing these regions, -131/+65 (P; distal) and +461/+675 (P'; proximal), were demonstrated in transient transfection experiments using reporter constructs containing the gene fragments linked individually upstream of the chloramphenicol acetyltransferase (CAT) gene. Cotransfection of P-CAT or P'-CAT constructs containing two upstream GAL4 binding sites into primary cultures of rat uterine cells with a vector expressing a GAL4 DNA binding domain-VP16 activating region fusion protein resulted in a 10-fold increase in CAT activity relative to cells transfected with either reporter and a vector expressing only the GAL4 DNA binding domain. The estrogen inducibility of the promoter-CAT constructs was assessed by transfection into MCF-7 breast cancer cells, which contain high levels of estrogen receptor (ER). P'-CAT, but not P-CAT, was induced by estradiol (E2; 8-fold). In primary rat uterine cells, which contain lower levels of ER, P'-CAT required the addition of one upstream consensus estrogen response element (ERE) to be estrogen inducible, whereas P-CAT required the addition of two EREs. Point and deletion mutants of the proximal promoter region in the P'-CAT reporter, screened in MCF-7 cells, were used to identify a 20-base pair fragment (+617/+636) that retained the promoter activity and 50% of the estrogen inducibility of P'. This fragment contained an ERE-like sequence conserved in 8 of 10 positions relative to the consensus ERE. Two copies of this sequence conferred estrogen inducibility (4-fold) when placed upstream of the distal promoter in P-CAT. To examine ER-dependent stimulation of the two PR gene promoters by cAMP, P-CAT and P'-CAT reporter constructs containing two upstream consensus EREs were cotransfected into ER-negative 3T3 cells with an ER expression vector. Induction by E2 was greater than 50-fold for both constructs. Treatment of the cells with agents that increase intracellular cAMP levels, namely cholera toxin plus isobutyl methylxanthine, resulted in CAT activity that was 8% and 51% of the E2-stimulated activity for the P and P' constructs, respectively.