The relationship between the colonization of Lotus japonicus by plant growth-promoting fungi (PGPF) and biosynthesis of the isoflavonoid phytoalexin vestitol, a major defensive response of leguminous plants, was analyzed. When PGPF including Trichoderma koningi, Fusarium equiseti, and Penicillium simplicissimum were inoculated onto L. japonicus roots, only T. koningi colonized the roots long-term and increased plant dry weight (126%). Microscopic observations of transverse sections of roots colonized by T. koningi demonstrated intercellular hyphal growth and the formation of yeast-like cells. The induction of plant defenses by fungal infections was examined by Northern analysis of genes involved in vestitol biosynthesis and HPLC of vestitol production in L. japonicus. Inoculation with symbiotic Mesorhizobium loti did not induce any accumulation of the transcripts. T. koningi immediately suppressed transcript levels to those induced by M. loti. The vestitol transuded from roots by T. koningi was detected at a level equivalent to that transuded by M. loti. Other PGPF and Calonectoria ilicola pathogenic to soybean but not to L. japonicus, stimulated continuous expression of genes and exudation of vestitol. These PGPF resembled mycorrhizal fungi in the establishment of symbiotic associations rather than fungal parasites.