Abstract Background: Prolactin is a lactogenic hormone associated with breast cancer risk in prospective studies, which used immunoassays. The immunoassay captures multiple isoforms and may not fully reflect the biologic activity of prolactin relevant to breast carcinogenesis. Methods: We considered plasma bioactive prolactin levels measured by the Nb2 lymphoma cell bioassay, which is sensitive to the somatolactogenic activity of prolactin and growth hormone, within a nested case–control study of invasive breast cancer in the Nurses' Health Studies (NHS/NHSII). We also considered associations with breast cancer risk factors. Results: We had bioassay measures on 1,329 cases and 1,329 controls. Bioassay levels were inversely associated with parity (4+ vs. 0 children = −18%, P = 0.01), body mass index (30+ vs. <22 kg/m2 = −16%, P < 0.01), and age at menopause (53+ vs. 48 years = −18%, P = 0.03) and positively with family history of breast cancer (yes vs. no = 14%, P < 0.01). The relative risk (RR) comparing the top versus bottom quartile of bioassay levels was 1.19 [95% confidence intervals (CI), 0.94–1.51; Ptrend = 0.18]. The association was suggestively stronger for postmenopausal (RR = 1.36; 95% CI, 0.93–1.98; Ptrend = 0.12) versus premenopausal women (RR = 0.99; 95% CI, 0.71–1.37; Ptrend = 0.71). There was an association for cases diagnosed <4 years after blood draw (RR = 2.66; 95% CI, 1.45–4.89; Ptrend < 0.01), but not for cases diagnosed later. We did not observe differential associations by estrogen receptor status or other tumor characteristics. Conclusions: Our results show similar associations for prolactin levels measured by bioassay and by immunoassay with both breast cancer risk factors and risk. Impact: Future work examining risk prediction model of breast cancer can use the immunoassay to accurately characterize risk. Cancer Epidemiol Biomarkers Prev; 24(1); 73–80. ©2014 AACR.