In the vicinity of big sagebrush (Artemisia tridentata), the growth of Nicotiana attenuata is negatively affected, in part due to the alleopathic effect of methyl jasmonate (MeJA) which is produced in large quantities by the aerial parts of sagebrush. Preliminary experiments suggested that growth-inhibiting substances were being emitted from the sagebrush roots. To identify the allelochemical secondary metabolites, we tested different root extracts in seedling growth bioassays with the naturally co-occurring native tobacco, Nicotiana attenuata, in a two-chamber Petri dish assay, optimized for tests of volatiles. Fractions rich in volatile compounds were particularly phytotoxic. We analyzed the volatiles emitted from the roots of intact Artemisia tridentata plants grown in soil, sand, and hydroponic cultures by using dynamic headspace extraction, headspace solvent-microextraction (HSME) and headspace solid-phase microextraction (HSPME), and GC-MS. Camphor, 1,8-cineol, nerol, and neryl isovalerate were phytotoxic and released as the major constituents. In addition to the phytotoxic monoterpenes, himachalenes, longifolene, caryophyllene, and acetylenic spiroethers, were found as characteristic components in the root's volatiles. The allelopathic potential of these root volatiles was compared with that of methyl jasmonate (MeJA), one of the most active compounds emitted from above-ground parts of the plant.