Ethylene inhibits the establishment of symbiosis between rhizobia and legumes. To examine how and when endogenous ethylene inhibits rhizobial infection and nodulation, we produced transgenic Lotus japonicus carrying the mutated melon ethylene receptor gene Cm-ERS1/H70A that confers ethylene insensitivity and fixes the transgene in the T(3) generation. The resultant transgenic plants showed reduced ethylene sensitivity because of 1-aminocyclopropane-1-carboxylate resistance and increased flowering duration, probably due to a dominant negative mechanism. When inoculated with Mesorhizobium loti, transgenic plants showed markedly higher numbers of infection threads and nodule primordia on their roots than did either wild-type or azygous plants during the early stage of cultivation period as well as during later stages, when the number of mature nodules had reached a steady state. In addition, transcripts of NIN, a gene governing infection thread formation, increased in the inoculated transgenic plants as compared with the wild-type plants. The infection responses of transgenic plants were similar to those of wild-type plants treated with ethylene inhibitors. These results imply that the endogenous ethylene in L. japonicus roots inhibits rhizobial infection at the primary nodulation, probably via NIN gene, and suggest that ethylene perception assists negative feedback regulation of secondary nodule initiation.