Exposure to light at night, as experienced by rotating night shift workers, has been related to lower circulating levels of melatonin, a hormone with recognized cancer protective properties. However, little is known about the relationship of other lifestyle factors or endogenous sex steroid hormones with melatonin levels. We examined cross-sectional associations of age, reproductive and menopausal factors, body mass index (BMI), alcohol consumption, smoking history, night shift work, as well as several other breast cancer risk factors, and circulating sex steroid hormone levels with creatinine-adjusted morning urinary melatonin (6-sulfatoxymelatonin, aMT6s) levels. Participants were 459 healthy, primarily premenopausal (age range 33-50 yr) women from the Nurses' Health Study II (NHS II). Using multiple linear regression, we computed least-square mean hormone levels across categories of lifestyle factors. Age was inversely related to aMT6s levels, particularly before menopause (premenopausal women, <or=39 yr versus >or=49 yr; aMT6s, 20.8 ng/mg versus 11.8 ng/mg creatinine; P for trend, 0.02). In multivariate analyses, BMI was significantly and inversely associated with aMT6s levels (P for trend, <0.01). Higher pack-years of smoking were associated with significantly lower aMT6s levels (never smoker versus 15+ pack-years, aMT6s = 17.4 ng/mg versus 12.3 ng/mg creatinine; P for trend, 0.04). We also observed a positive association between parity and aMT6s levels (P for trend, <0.01), but no other reproductive factors nor any of the sex hormones (estradiol, progesterone, estrone, estrone sulfate, dehydroepiandrostenedione, dehydroepiandrostenedione sulfate, testosterone, and androstenedione), as measured either in the luteal or the follicular phase of the menstrual cycle, were significantly associated with aMT6s. In conclusion, higher age, BMI, and heavy smoking were significantly related to lower levels of melatonin, whereas parity was significantly associated with higher aMT6s levels. Melatonin levels may be one mechanism through which these factors influence the development of cancer, but more studies are needed to elucidate these mechanisms definitively.