The objective of this study was to evaluate the possibility of selecting genetic variants of plants with enhanced concentrations of the indoleamine melatonin. A germplasm line of the medicinal plant species, St John's wort (Hypericum perforatum L.), with high levels of melatonin was selected in vitro using mutagenized tissues. The germplasm line has remained stable over a 5-yr period and contained >12-fold (1200%) melatonin content compared with the wild-type plant. Melatonin is a ubiquitous, highly conserved molecule with known therapeutic roles in the treatment of sleep disorders, depression, aging, inhibition of cancer cell growth and as a free radical scavenger and antioxidant. The selected melatonin-rich germplasm line of St John's wort may facilitate fundamental studies on melatonin biosynthesis, metabolism and new developments in natural products for treatment of human diseases.