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Melatonin shows potential oncostatic activity and is acutely suppressed by light exposure. Some evidence suggests an association between night work and breast cancer risk, possibly through the melatonin pathway.In a cohort of premenopausal nurses, we prospectively studied the relation between rotating night shift work and breast cancer risk. Total number of months during which the nurses worked rotating night shifts was first assessed at baseline in 1989 and periodically updated thereafter. We used Cox proportional hazards models to calculate relative risks (RRs) and 95% confidence intervals (CIs).Among 115,022 women without cancer at baseline, 1,352 developed invasive breast cancer during 12 years of follow up. Women who reported more than 20 years of rotating night shift work experienced an elevated relative risk of breast cancer compared with women who did not report any rotating night shift work (multivariate RR = 1.79; 95% CI = 1.06–3.01). There was no increase in risk associated with fewer years of rotating night work.Our results suggest a modestly elevated risk of breast cancer after longer periods of rotating night work. Additional studies are warranted to rule out small sample size or uncontrolled sources for confounding as alternative explanations.