Abstract.To identify bacterial genes involved in symbiotic nodule development, ineffective nodules of alfalfa (Medicago sativa) induced by 64 different Fixmutants of Rhizobium meliloti were characterized by assaying for symbiotic gene expression and by morphological studies. The expression of leghemoglobin and nodulin-25 genes from alfalfa and of the nifHD genes from R. meliloti were monitored by hybridizing the appropriate DNA probes to RNA samples prepared from nodules. The mutants were accordingly divided into three groups. In group I none of the genes were expressed, in group II only the plant genes were expressed and in group III all three genes were transcribed. Light and electron microscopical analysis of nodules revealed that nodule development was halted at different stages in nodules induced by different group I mutants. In most cases nodules were empty lacking infection threads and bacteroids or nodules contained infection threads and a few released bacteroids. In nodules induced by a third mutant class bacteria were released into the host cells, however the formation of the peribacteroid membrane was not normal. On this basis we suggest that peribacteroid membrane formation precedes leghemoglobin and nodulin-25 induction, moreover, after induction of nodulation by the nod genes at least two communication steps between the bacteria and the host plants are necessary for the development of the mature nodule. By complementing each mutant of group I with a genomic R. meliloti library made in pLAFR1, four new fix loci were identified, indicating that several bacterial genes are involved in late nodule development.HIZOBIA are able to cooperate with leguminous plants to fix atmospheric nitrogen. Nitrogen fixation takes place in symbiotic nodules, new plant organs developed for this purpose. Nodule cells harbor unique cell organelles, bacteroids, derived from Rhizobium bacteria which reduce dinitrogen to ammonia. The differentiation and function of nodules involves symbiosis-specific expression of both bacterial and plant genes (nodulin genes) in a highly coordinated manner, as well as communication between the two partners. This communication process seems to consist of signal exchange during nodule development. The molecular basis of these events are, at present, mostly unknown.Recently, plant flavones were reported to act as signal molecules inducing the expression of bacterial nodulation (nod)