Ailanthone is an allelopathic chemical that is produced by the Ailanthus altissima tree which inhibits the growth of other plants. Ailanthone-rich fractions were obtained from A. altissima bark by extraction with dichloromethane and ethyl acetate and subsequent purification of these crude extracts, and of the remaining water mixture after solvent extraction, by means of gel permeation chromatography. A number of fractions were isolated and characterized for ailanthone content. A significant pre-emergence herbicide activity was found for most of the extracts which was directly correlated to ailanthone concentration. A remarkable combined pre- and post-emergence herbicide activity was found for a specific fraction. These results indicate that the bark of A. altissima may represent an interesting source for the production of natural herbicides for use in agriculture.