CS2 is a volatile liquid with bacteriostatic, fungicidal, nematicidal and insecticidal properties. The roots of some plants, particularly those in the subfamily Mimosoideae, produce CS2. These plants may have an agronomic use in crop rotations or intercropping because root-produced CS2 may act as a deterrent to soil pathogens. The biochemical pathway leading to the production of CS2 from mimosoid roots is unclear. Presumably, S-alkyl cysteine lyases present in mimosoid seedlings hydrolyze L-djenkolic acid, a unique, non-protein, sulfur-containing amino acid, to pyruvate, ammonia and methylene dithiol. Because methylene dithiol has never been detected from mimosoid roots, we hypothesized that CS2 is produced instead. Mimosa pudica, a species which produces CS2 in its roots, was used as a model plant. To eliminate CS2 production from microbial sources, all plants were grown gnotobiotically. S-alkyl cysteine lyase activity was confirmed in M. pudica when PbS formed on injured roots treated with L-cysteine and lead acetate. When injured roots were wetted, CS2 production increased significantly compared to non-wetted roots. When L-djenkolic was applied to injured roots, CS2 production increased significantly compared to controls. Both thin-layer paper chromatography and high performance liquid chromatography showed the presence of L-djenkolic acid in root tissues of M. pudica. These findings suggest CS2 production in roots of M. pudica occurs via the hydrolysis of L-djenkolic acid by S-alkyl cysteine lyase.