Background: Experimental and epidemiological data suggest that higher circulating prolactin is associated with breast cancer risk; however how various risk factors for breast cancer influence prolactin levels in healthy women is not clear. Methods: We analysed cross-sectional associations between several suggested reproductive and lifestyle risk factors for breast cancer and circulating prolactin among pre- and postmenopausal women, taking into account the use of current postmenopausal hormone therapy (HT), among 2560 controls from a breast cancer nested case-control study within the EPIC cohort. Results: Adjusted geometric mean prolactin levels were significantly higher among premenopausal women, and among postmenopausal women using HT compared to non-users (8.2, 7.0 and 6.3 ng/ml, respectively, Pcat=<0.0001). Furthermore, prolactin levels were significantly higher among users of combined estrogen-progestin HT compared to users of estrogen-alone HT (6.66 vs 5.90 ng/ml, Pcat=0.001). Prolactin levels were lower among parous women compared to nulliparous women (8.61 vs 10.95 ng/ml, Pcat=0.0002, premenopausal women); the magnitude of this difference depended on the number of full-term-pregnancies (22.1% lower, ≥3 vs 1 pregnancy, Ptrend=0.01). Results for parity were similar but lower in magnitude among postmenopausal women. Prolactin did not vary by other studied factors, with the exception of lower levels among postmenopausal smokers compared to never-smokers. Conclusions: Our study shows that current HT use, especially the use of combined HT, is associated with higher circulating prolactin levels in postmenopausal women, and confirms prior findings of lower prolactin in parous women. Impact: Our study extends the knowledge linking various breast cancer risk factors with circulating prolactin.