Recent evidence suggests that prolactin may be positively associated with postmenopausal breast cancer risk; however, little data are available in younger women. Therefore, we conducted a prospective, nested case-control study to examine the relationship between plasma prolactin concentrations and breast cancer risk in predominately premenopausal women from the Nurses' Health Study II. Blood samples were collected from 1996 to 1999. The analysis includes 316 cases of breast cancer diagnosed after blood donation and before June 1, 2003, who had two controls matched on age, fasting status, time of day and month of blood collection, race/ethnicity, and timing of blood draw within the menstrual cycle. Sixty-three percent of participants provided a timed follicular and luteal menstrual phase blood sample; other women provided a single untimed sample. When including all women, we observed a positive association between prolactin and breast cancer risk [relative risk (RR), top quartile versus bottom quartile, 1.5; 95% confidence interval (95% CI), 1.0-2.3; P(trend) = 0.03] that was slightly stronger among estrogen receptor-positive/progesterone receptor-positive tumors (comparable RR, 1.9; 95% CI, 1.1-3.3; P(trend) = 0.04). Associations were similar among premenopausal women only. However, we did not find an association between prolactin and breast cancer risk among the subset of women who only provided timed samples (comparable RR, average of timed samples, 1.3; 95% CI, 0.8-2.3; P(trend) = 0.40). The association seemed stronger among women > or = 45 years old and for cases diagnosed within approximately 4 years of blood collection. Our data suggest a modest positive association between prolactin and breast cancer risk among predominately premenopausal women; however, further follow-up is needed to increase power for subgroup analyses.