The progesterone receptor (PR) and its coactivators and corepressors play an important role in female reproductive function. To investigate the functional interactions between PR and steroid receptor coactivators (SRCs) required for regulation of gene transcription in vivo, we crossed PR activity indicator (PRAI) mice with SRC-1^(+/−) and SRC-3^(+/−) mice to generate bigenic mice, PRAI-SRC-1^(−/−) and PRAI-SRC-3^(−/−). In the mammary gland, PR activity in the luminal epithelium of both wild-type and SRC-1^(−/−) mice was induced by estrogen + progesterone treatment. In contrast, an increase in PR activity in the luminal epithelium was not detected in SRC-3^(−/−) mice with the same treatment. In the uterus, PR activity in the stroma compartment of both wild-type and SRC-3^(−/−) mice was induced by estrogen + progesterone treatment. However, the increased PR activity was not detected in SRC-1^(−/−) mice. Taken together, our data indicate that the endogenous physiological function of PR in distinct tissues is modulated by different steroid receptor coregulators. SRC-3 is the primary coactivator for PR in breast and SRC-1 is the primary coactivator for PR in uterus.