A growing literature indicates that blood levels of the hormone melatonin may have important implications for human health and well-being. Melatonin is synthesized and released into the general circulation at night, however, and it is seldom feasible to draw blood samples at night in epidemiological studies. There is some evidence that levels of urinary melatonin and of 6-sulfatoxymelatonin (aMT6s), the major metabolite of melatonin, accurately reflect nocturnal plasma melatonin. If this is the case, urinary assays could be powerful tools for epidemiological studies. A laboratory-based study was performed to examine the relationships between nocturnal plasma melatonin, morning urinary melatonin, and morning urinary aMT6s levels in 78 men. The relationship between total nocturnal plasma melatonin and both urinary aMT6s corrected for creatinine and urinary melatonin is significant. Combining the two urinary measures accounts for 72% of the variance in total plasma melatonin. Peak nocturnal plasma melatonin also was significantly related to urinary melatonin and to aMT6s. The urinary measures show good sensitivity and specificity in identifying individual differences in nocturnal plasma melatonin levels. These results support the inclusion of morning urine samples to assess the contribution of the hormone melatonin in occupational or residential studies involving healthy, young men.