To define the functional relevance of progesterone-initiated intracellular signaling in mammary gland tumorigenesis, the progesterone receptor knockout (PRKO) mouse model was used in the context of an established carcinogen-induced mammary tumorigenesis system. In carcinogen-treated, 7,12-dimethylbenz(a)anthracene (DMBA), pituitary-isografted mice, there was a marked reduction in mammary tumor incidence in PRKO mice as compared with isogenic wild types (WT). Mammary tumors arose in 12 (60%) of 20 WT mice compared with 3 (15%) of 20 PRKO mice by 44 weeks after the initial DMBA treatment. In the absence of a pituitary isograft, mammary tumors developed in 4 (20%) of 20 WT mice versus 4 (20%) of 20 PRKO mice by 47 weeks. At the time of carcinogen administration, the proliferative index of the pituitary-stimulated WT gland was at least 4-fold higher than similarly treated PRKO glands, supporting the importance of PR-mediated proliferative pathways in the genesis of this tumor type. Unlike the WT gland, the PRKO gland was unable to exhibit alveologenesis in response to pituitary isograft stimulation; thus, DMBA-initiated mammary tumors observed in the PRKO were assumed to be exclusively of ductal origin. Compared with previous tested strains, by 47 weeks, a higher incidence of DMBA-induced ovarian tumors was observed in this mouse strain: (a) 4 (20%) of 20 WT mice and 9 (45%) of 20 PRKO mice with a pituitary isograft; and (b) 10 (50%) of 20 WT mice and 10 (50%) of 20 PRKO mice without a pituitary isograft. Despite the host-strain's underlying propensity for DMBA-induced ovarian neoplasms, our studies underscore the specific importance of the PR (as distinct from the estrogen receptor) as a mandatory mediator for those intracellular signaling pathways that are essential for the initiation of the majority of murine mammary tumors induced by DMBA. Apart from providing strong support for progesterone's role in mammary gland tumorigenesis as well as furthering our fundamental understanding of breast cancer etiology, these studies may have implications for the routine use of progestins.