RU486 is an incomplete progesterone receptor (PR) antagonist due to its partial agonist activity. To investigate the tissue-specific effects of RU486 on PR function in an ovariectomized mouse model, we used the progesterone receptor activity indicator mouse and evaluated the key determinants of progesterone-dependent gene activity: PR, coregulators, and kinases. As might be expected, acute RU486 treatment (6 h after injection) reduced PR transcriptional activity in the uterus, compared with vehicle or progesterone (P4) treatments. Chronic RU486 treatment (3 d) had a distinctly different effect on PR-mediated transcription, elevating PR activity in both the uterus and mammary gland, whereas chronic P4 treatment reduced PR activity in both tissues. This elevated uterine PR activity was associated with increased modified forms of PR and total protein levels of steroid receptor coactivator (SRC)-1 without affecting SRC-2 or SRC-3 protein levels. In addition to increased levels of coactivators, chronic RU486 treatment activated the ERK-1/2 and c-Jun N-terminal kinase signaling pathways in the uterus in a manner comparable with P4 treatment. In contrast to our observations in the uterus, chronic RU486 treatment increased modified forms of PR and the SRC-3 protein levels (but not SRC-1 and SRC-2 levels) in luminal epithelial cells of the mammary gland. Chronic RU486 also activated the c-Jun N-terminal kinase, but not ERK-1/2, signaling pathways in mammary luminal epithelial cells. This report suggests that in comparison with chronic natural hormone (P4), a mixed antagonist/agonist (RU486) induces a distinct temporal and spatial pattern of cellular genetic regulators that accompany ligand-specific gene expression.