BACKGROUND:Breast cancer (BCa) mortality is decreasing with early detection and improvement in therapies. The incidence of BCa, however, continues to increase, particularly estrogen-receptor-positive (ER +) subtypes. One of the greatest modifiers of ER + BCa risk is childbearing (parity), with BCa risk halved in young multiparous mothers. Despite convincing epidemiological data, the biology that underpins this protection remains unclear. Parity-induced protection has been postulated to be due to a decrease in mammary stem cells (MaSCs); however, reports to date have provided conflicting data. METHODS:We have completed rigorous functional testing of repopulating activity in parous mice using unfractionated and MaSC (CD24midCD49fhi)-enriched populations. We also developed a novel serial transplant method to enable us to assess self-renewal of MaSC following pregnancy. Lastly, as each pregnancy confers additional BCa protection, we subjected mice to multiple rounds of pregnancy to assess whether additional pregnancies impact MaSC activity. RESULTS:Here, we report that while repopulating activity in the mammary gland is reduced by parity in the unfractionated gland, it is not due to a loss in the classically defined MaSC (CD24+CD49fhi) numbers or function. Self-renewal was unaffected by parity and additional rounds of pregnancy also did not lead to a decrease in MaSC activity. CONCLUSIONS:Our data show instead that parity impacts on the stem-like activity of cells outside the MaSC population.