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Melatonin, a pineal hormone present in the blood of humans and other species, has a distinct diurnal variation in its biosynthesis and, therefore, in its concentration. This variation has suggested the possibility of a regulatory function in day/night-dependent physiological processes such as sleep and has led scientists to explore the effects of administered melatonin on the modulation of circadian rhythms. For the self-treatment of sleep disorders and other benefits, melatonin use has been extolled to the extent that 20 million new consumers were added to the U.S. retail market in 1995. Its principal aeromedical application has been in the experimental treatment of jet-lag effects. For aircraft passengers, melatonin administration at destination bedtime appears to improve sleep quality and to decrease the time required to reestablish normal circadian rhythms. For international aircrews that travel through multiple time zones without time to adapt to new environments, taking melatonin before arriving home may further impair already disturbed circadian rhythms. Its use to adjust to shiftwork changes by air traffic controllers, aircraft maintenance workers, and support personnel is even more controversial. Limited studies suggest that giving this hormone to shift workers should be done only under controlled conditions and that taking it at the wrong time may actually impair job performance. Because of its possible interaction with certain medications and the changes in its concentrations observed in some clinical conditions, the practitioner must exercise caution during the medical certification of airmen. The variations in the concentration of melatonin can be effectively determined by radioimmunoassay, high-performance liquid chromatography, and gas chromatography-mass spectroscopy analytical techniques. These techniques are capable of measuring the human daytime (10 pg/mL) and nighttime (30–120 pg/mL) melatonin in plasma/serum. Melatonin measurements in victims of accidental death may allow forensic scientists and accident investigators to use the relationship between its concentration and the time of day when death occurred. The most accurate estimations of the time of death result from analysis of melatonin content of the whole pineal body, whereas less accurate estimates are obtained from serum and urine analyses. Pineal levels of melatonin are unlikely to be altered by exogenous melatonin, but its blood and urine levels would change. High blood levels in a daytime crash victim would suggest exogenous supplementation. The possible interfering effects of postmortem biochemical processes on melatonin concentrations in whole blood and in other tissues are not well understood, and there is a need for the continuing research into melatonins chronobiological properties to define its proper applications and limitations. The indiscriminate use of melatonin by aviation professionals may pose unacceptable safety risks for air travel.