BACKGROUND: We investigated the association between oral contraceptives (OCs) and CD44, CD24, and ALDH1A1 stem cell (SC) markers' expression in benign breast tissue. METHODS: We included 731 cancer-free women with biopsy-confirmed benign breast disease (BBD) within the Nurses' Health Study II. Information on OC use was collected in 1989 and updated biennially. Immunohistochemistry (IHC) was performed on tissue microarrays constructed from histologically normal breast tissue cores. For each core, the IHC expression was assessed with semi-automated software as the % of positively stained cells for each marker. Generalized linear regression was used to examine the associations of OCs (use status, total duration, age at first and last use, use before first pregnancy, and time since last use) with each marker's log-transformed expression (within stroma and epithelium). RESULTS: In multivariate analyses, OC use was positively associated with stromal CD44 expression (current vs. never β = 0.75, 95% Confidence Interval [CI] 0.11, 1.39, p-trend = 0.02). Age at first use was inversely associated with CD44 expression (in stroma: β= -0.04, 95% CI -0.08, -0.01; in epithelium β= -0.04, 95% CI -0.06, -0.02). Use before first pregnancy was positively associated with CD44 in epithelium (β = 0.43; 95% CI 0.13, 0.74). Total duration, age at last use, and time since last use were not associated with any of the SC markers. CONCLUSIONS: Our findings suggest a greater expression of CD44 in the breast stroma of current OC users and a reduced CD44 expression with a later age at first OC use, consistent with the reported associations of OCs with BCa risk.