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 employed the Progesterone Receptor Activity Indicator (PRAI) mouse and evaluated the key determinants of progesterone-dependant 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 to vehicle or progesterone (P4) treatments. Chronic RU486 treatment (3 days) had a distinctly different effect on PR-mediated transcription, elevating PR activity in both the uterus and mammary gland, while 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 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 JNK 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 JNK, but not ERK-1/2, signaling pathways in mammary luminal epithelial cells. This report suggests that in comparison to chronic natural hormone (progesterone), a mixed antagonist/agonist (RU486) induces a distinct temporal and spatial pattern of cellular genetic regulators that accompany ligand specific gene expression.