The progesterone receptor (PR) gene is activated by estrogen in normal reproductive tissues and in MCF-7 human breast cancer cells. Although it is typically thought that estrogen responsiveness is mediated through estrogen response elements (EREs), the human PR gene lacks a palindromic ERE sequence. We have identified an activating protein-1 (AP-1) site at +745 in the human PR gene that bound purified Fos and Jun and formed a complex with Fos/Jun heterodimers present in MCF-7 nuclear extracts. Surprisingly, mutating the +745 AP-1 site in the context of a 1.5-kb region of the PR gene significantly enhanced estrogen receptor (ER) alpha-mediated transactivation, suggesting that the wild-type +745 AP-1 site plays a role in inhibiting PR gene expression in the presence of hormone. In support of this idea, transient transfection assays demonstrated that increasing levels of Fos and Jun repressed transcription of a reporter plasmid containing the +745 AP-1 site. Fos levels were transiently increased, ERalpha levels were decreased, and Jun was dephosphorylated after MCF-7 cells were treated with estrogen. Chromatin immunoprecipitation assays demonstrated that Jun was associated with the +745 AP-1 site in the endogenous PR gene in the presence and in the absence of estrogen, but that ERalpha and Fos were only associated with the +745 AP-1 site after estrogen treatment of MCF-7 cells. Our studies suggest that the human PR gene is regulated by multiple transcription factors and that the differential binding of these dynamically regulated trans-acting factors influences gene expression.