Tetronic acid is a candidate for an inducer of nodulation (nod) gene expression in Mesorhizobium loti. However, there is no evidence that tetronic acid is secreted from Lotus species, the host plants of M. loti. This suggests that Lotus plants may produce other genuine nod gene inducers that differ from tetronic acid. In this study, M. loti cells harboring a nodA-lacZ fusion were enclosed in dialysis tubing and immersed in L. corniculatus culture solution. Significant β-galactosidase activities were detected in the M. loti cells, indicating that nod gene inducers were present in the seed and root exudates. β-galactosidase activities in M. loti cells were also determined after cells were exposed to seed and root exudates or tetronic acid in vitro. Seed and root exudates induced greater β-galactosidase activity in M. loti cells in 1 mM CaSO_4 solution than in YEM medium. In contrast, tetronic acid induced greater β-galactosidase activity in M. loti cells in YEM medium than in 1 mM CaSO_4 solution. Further, β-galactosidase activities induced by seed and root exudates and tetronic acid tended to decrease as the pH of the medium became neutral. In an LC-MS analysis, we could not detect tetronic acid in the concentrated seed and root exudates. These findings indicate that Lotus plants release genuine nod gene inducers for M. loti that differ from tetronic acid. Finally, seed exudates of L. corniculatus were fractionated according to molecular size and polarity, and it was clarified that a putative nod gene inducer in the seed exudates was a low molecular weight and high polarity substance.