Understanding how the timing of exposure to endogenous hormones influences cancer development is critical to elucidating disease etiology. Prolactin increases proliferation and cell motility, processes important in later stage tumor development, suggesting that levels proximate (versus distant) to diagnosis may better predict risk. Thus, we calculated relative risks (RR) and 95% confidence intervals (CI) for prolactin levels on samples collected <10 (proximate) versus ≥10 (distant) years before diagnosis in the Nurses' Health Study (NHS) and NHSII with breast cancer risk, including in a subset of NHS women providing two samples 10 years apart. We measured prolactin via immunoassay in cases diagnosed from 1990 to 2010 (NHS) and 1999 to 2009 (NHSII) and matched controls. Overall, 2,468 cases and 4,021 controls had prolactin measured <10 years and 953 cases and 1,339 controls >10 years before diagnosis/reference date. There was an increased risk for higher proximate prolactin levels [RR, >15.7 vs. ≤8.1 ng/mL (i.e., top vs. bottom quartiles) = 1.20; 95% CI, 1.03-1.40; Ptrend = 0.005], but not for distant levels (RR = 0.97; Ptrend = 0.94); results were similar among women with two blood samples (Pinteraction, proximate vs. distant = 0.07). The positive association was stronger for ER(+) disease (RR = 1.28; Ptrend = 0.003) and postmenopausal women (RR = 1.37; Ptrend = 0.0002). Among postmenopausal women, the association was strongest for ER(+) disease (RR = 1.52) and lymph node-positive cases (RR = 1.63). Our data suggest that prolactin levels measured <10 years before diagnosis are most strongly associated with postmenopausal breast cancer risk, especially for ER(+) tumors and metastatic disease. This corresponds with biologic data that prolactin is etiologically important in tumor promotion.