||BACKGROUND: An increased risk of breast cancer has been observed in night shift workers. Exposure to artificial light at night and disruption of the endogenous circadian rhythm with suppression of the melatonin synthesis have been suggested mechanisms. We investigated the hypothesis that rotating night shift work is associated with mammographic density. METHODS: We conducted a cross-sectional study on the association between rotating night shift work characteristics, 6-sulfatoxymelatonin (MT6s) creatinine adjusted in a spot morning urine sample, and a computer-assisted measure of mammographic density in 640 nurses and midwives ages 40 to 60 years. The associations were evaluated using regression models adjusted for age, body mass index, menopausal status, age at menopause, age at menarche, smoking, and the calendar season of the year when mammography was conducted. RESULTS: The adjusted means of percentage of mammographic density and absolute density were slightly higher among women working rotating night shifts but not statistically significant [percentage of mammographic density = 23.6%, 95% confidence interval (CI), 21.9%-25.4% vs. 22.5%, 95% CI, 20.8%-24.3%; absolute density = 23.9 cm(2), 95% CI, 21.4-26.4 cm(2) vs. 21.8 cm(2), 95% CI, 19.4-24.3 cm(2) in rotating night shift and day shift nurses, respectively). There were no significant associations between the current or cumulative rotating night shift work exposure metrics and mammographic density. No association was observed between morning MT6s and mammographic density. CONCLUSIONS: The hypothesis on the link between rotating night shift work, melatonin synthesis disruption, and mammographic density is not supported by the results of the present study. IMPACT: It is unlikely that the development of breast cancer in nurses working rotating night shifts is mediated by an increase in mammographic density.