The root parasitic plant, Striga hermonthica, constrains the production of several agronomically important poaceous crops in the arid and semiarid tropical regions of Sub-Saharan Africa. The parasite is incompatible with the model legume, Lotus japonicus. Studies at the molecular and metabolic levels have revealed that expression of the genes involved in the biosynthesis of vestitol, a legume-specific phytoalexin, was highly up-regulated in L. japonicus roots challenged with S. hermonthica. High-performance liquid chromatography and mass spectroscopy confirmed the presence of vestitol in the exudate released from L. japonicus roots inoculated with S. hermonthica seedlings. Fluorescence, similar to that emitted by authentic vestitol, was displayed on the surface of L. japonicus roots to which successful attachment of S. hermonthica had been achieved. Vestitol exerted a limited inhibitory effect on S. hermonthica germination, but it significantly inhibited seedling growth. These results indicate that vestitol biosynthesis in L. japonicus was induced by S. hermonthica attachment and that vestitol contributed, at least in part, to the host's defence mechanism and acted as a chemical barrier against the intrusion of the parasite.