Purpose Epidemiologic studies suggest that prolactin is associated with breast cancer risk in older women. Because of limited prospective data, particularly in younger women, we examined whether prolactin concentrations were associated with breast cancer risk among women 42 to 55 years (68% premenopausal) from the Nurses’ Health Study (NHS), and then conducted a pooled analysis of three studies. Patients and Methods The analysis included 377 cases of breast cancer diagnosed after blood draw and before June 2000; two controls were matched per case on age, menopausal status at blood draw and diagnosis, fasting status, and time of day and month of blood collection. These data were pooled with two previously published data sets from the NHS and NHSII cohorts (n = 1,539 cases, 2,681 controls; ages 32 to 70 years). Results Prolactin was modestly associated with an increased breast cancer risk (relative risk [RR], top v bottom quartile = 1.3; 95% CI, 0.9 to 1.9; P for trend = .12). Risk estimates did not vary by menopausal status, tumor invasiveness, or estrogen receptor (ER) status. In the pooled analysis, the overall RR was 1.3 (95% CI, 1.1 to 1.6; P for trend = .002), and did not vary by menopausal status (P for interaction = .95). The risk was strongest for women with ER+ tumors (RR = 1.6; 95% CI, 1.2 to 2.0; P for trend < .001). Correcting for within-person variability, the RR comparing the median of the top versus the bottom prolactin quartile increased from 1.3 to 1.7 for all women and from 1.5 to 2.1 for ER+ cases. Conclusion These data, in conjunction with experimental studies, indicate that prolactin likely is important in breast cancer etiology, particularly ER+ tumors, over a wide range of ages.