We report the identification of new molecular markers associated with different stages of Rhizobium-induced nodule development in the legume Medicago truncatula. A cDNA library was constructed from pre-nitrogen-fixing M. Truncatula nodules, and differentially screened with a polymerase chain reaction-amplified subtracted probe. Twenty-nine new families of nodulin cDNA clones, designated MtN1 to MtN29, were thus identified in addition to clones for several known nodulins. All MtN genes were shown by Northern (RNA) hybridization analysis to be induced during nodulation, some of them well before nodule emergence. The MtN genes were classified into three groups depending on their expression kinetics. The expression of three MtN genes showed a limited induction by Nod factors purified from Rhizobium meliloti. Homologies with a variety of proteins were found for the deduced amino acid sequences of 10 of the MtN genes.