Women exposed to exogenous progesterone have increased breast cancer risk, but the mechanisms of progesterone involvement in breast cancer development are unknown. In human breast and endometrium, progesterone receptor (PR) isoform expression is disrupted in premalignant lesions and predominance of one isoform, usually PRA, in invasive cancers is associated with poorer prognosis. Disrupted PR isoform expression results in disrupted progestin regulation of cell morphology, including rounded morphology and decreased adherence of cells to tissue culture flasks. The purpose of this study was to test the hypothesis that predominance of PRA affects the interaction of breast cancer cells with a physiologically relevant stromal tissue, bone marrow stroma. T-47D breast cancer cells demonstrated the ability to migrate into bone marrow fibroblasts and this was inhibited by progestin treatment. The antiprogestin RU38486 abrogated the progestin effect on migration, demonstrating that it was PR-mediated. In cells expressing a predominance of PRA, after induction of a stably integrated inducible PRA construct, the ability of progestin to inhibit breast cancer cell migration was lost. A number of integrins were progestin regulated in T-47D cells, but there was no difference in the progestin effect in cells with PRA predominance, nor were the levels of focal adhesion proteins altered in these cells. This suggested that the lack of inhibition by progestin of breast cancer cell migration in cells with PRA predominance was not mediated by PRA effects on the membrane components of the adherens junctions. In summary, this study has shown that PRA predominance has a striking functional effect on breast cancer cell migration into stromal layers. PRA predominance may render breast cancer cells relatively resistant to the inhibitory effects of progestins and one consequence of this may be increased invasion of stroma. If borne out in vivo, these findings suggest that tumours with PRA predominance may be predisposed to cancer progression and this may signal a poorer prognosis in patients.