Four genes encoding small proteins with significantly high glycine content have been identified from root nodules of Medicago sativa. All of these proteins as well as their Medicago truncatula homologues carried an amino terminal signal peptide and a glycine-rich carboxy terminal domain. All except nodGRP3 lacked the characteristic repeat structure described for cell wall and stress response-related glycine-rich proteins (GRP). Expression of these GRP genes was undetectable in flower, leaf, stem, and hypocotyl cells, whereas expression was highly induced during root nodule development, suggesting that GRP genes act as nodulins. Moreover, none of these nodule-expressed GRP genes were activated by hormones or stress treatments, which are inducers of many other GRPs. In Rhizobium-free spontaneous nodules and in nodules induced by a noninfective mutant strain of Sinorhizobium meliloti, all these genes were repressed, while they were induced in Fix- nodules, unaffected in bacterial infection, but halted in bacteroid differentiation. These results demonstrated that bacterial infection but not bacteroid differentiation is required for the induction of the nodule-specific GRP genes. Differences in kinetics and localization of gene activation as well as in the primary structure of proteins suggest nonredundant roles for these GRPs in nodule organogenesis.