Prolactin is important in human breast development, and substantial laboratory and in vitro data suggest a role in mammary carcinogenesis. Therefore, we conducted a prospective case-control study nested within the Nurses' Health Study cohort to examine, in detail, the association between plasma prolactin concentrations and postmenopausal breast cancer by cancer invasiveness, estrogen receptor/progesterone receptor status, and other subject characteristics, including postmenopausal hormone use. Blood samples were collected from 1989 to 1990 and prolactin was measured by microparticle enzyme immunoassay. The analysis included 851 cases of postmenopausal breast cancer diagnosed after blood collection and before June 2000, in which there were one or two controls (n=1,275) matched on age, postmenopausal hormone use, fasting status, and time of day and month of blood collection. Prolactin was associated with a modestly increased risk of postmenopausal breast cancer [relative risk, top versus bottom quartile, 1.34; 95% confidence interval (CI), 1.02-1.76; P-trend = 0.01]. The association differed by estrogen receptor/progesterone receptor status (P-heterogeneity=0.03). The relative risk was 1.78 (95% CI, 1.28, 2.50; P-trend < 0.001) for estrogen receptor+/progesterone receptor+, 0.76 (95% CI, 0.43, 1.32; P-trend=0.28) for estrogen receptor-/progesterone receptor-, and 1.94 (95% CI, 0.99, 3.78; P-trend=0.12) for estrogen receptor+/progesterone receptor- breast cancers. Associations generally were similar for ductal and lobular carcinomas (P-heterogeneity=0.43) and by tumor size (P-heterogeneity=0.24). Among estrogen receptor+/progesterone receptor+ cancers, the association did not significantly differ by postmenopausal hormone use, years between blood draw and diagnosis, or after adjustment for estradiol (relative risk, 1.93; 95% CI, 1.16, 3.22; P-trend=0.01). Our prospective data suggest that plasma prolactin concentrations are associated with an increased risk of postmenopausal breast cancer, particularly for estrogen receptor+/progesterone receptor+ cancers, and independently of estradiol.