To clarify the allelopathic system of lemon balm (Melissa officinalisL.), purification and isolation of allelopathic substances was undertaken in acetone extract of the shoots of the 30-d-old plants. Three growth-inhibiting substances were found in the neutral fraction of the extract after silica gel column chromatography, and most active substance was further purified and isolated by thin layer chromatography, C18Sep Pack cartridge and reversephase high performance liquid chromatography. This substance inhibited the growth of cress (Lepidium sativunL.) at concentrations greater than 0.3 μg mL-1and the concentration required for 50% growth inhibition was 2.9μg mL-1. These results suggest that lemon balm shoots may contain at least three growth-inhibiting substances, which may be released into the environment, either as exudates from living tissues or leachates from residues of the plant, and may act as allelochemicals to neighboring plants.