Soluble factors released from roots of the pre-mycorrhizal infection (pmi) myc(-) tomato mutant M161 were analyzed and compared with normal wild-type released factors. Aseptic whole exudates from the M161 mutant retarded the proliferation of Glomus intraradices in vitro. When the whole exudate was further fractionated on a C18 SEPAK cartridge, the 50/70% methanol fraction showed an activity against hyphal tip growth of Gigaspora gigantea and Gl. intraradices. Preliminary characterization of the exudate suggests that the inhibitory moieties are heat labile, bind to PVPP (polyvinyl polypyrrolidone), and are not volatile. This is the first reported instance of the inhibition by a myc(-) plant being ascribed to inhibitory component(s) released in root exudate.