Abstract: The presence of melatonin (N-acetyl-5-methoxytryptamine) in plants has been clearly demonstrated. However, while this indoleamine has been intensively studied in animals, especially in mammals, the same is not true in the case of plants, where one of the most interesting aspects is its possible role as antioxidative molecule in physiological processes. Some data reflect the possible protective role that melatonin may exert in some stress situations such as ultraviolet (UV)-radiation, induced senescence and copper stress. The present work was designed to establish how the melatonin content changes in plants as a result of chemically induced stress. For this, barley plants were exposed in different treatments to the chemical-stress agents: sodium chloride, zinc sulphate or hydrogen peroxide. After different times, the content of melatonin in treated roots and control roots were determined using liquid chromatography (LC) with time-of-flight/mass spectrometry and LC with fluorescence detection for identification and quantification, respectively. The data show that the melatonin content in roots increased due to stress, reaching up to six times the melatonin content of control roots. Induction was time dependent, while hydrogen peroxide (10 mm) and zinc sulphate (1 mm) were the most effective inducers. The capacity of roots to absorb melatonin from soil was also studied. The data establish, for first time, that the chemical-stress agents assayed can induce the biosynthesis of melatonin in barley roots and produce a significant increase in their melatonin content. Such an increase in melatonin probably plays an important antioxidative role in the defense against chemically induced stress and other abiotic/biotic stresses.