Sinorhizobium meliloti enters an endosymbiosis with alfalfa plants through the formation of nitrogen-fixing nodules. In order to identify S. meliloti genes required for symbiosis and competitiveness, a method of signature-tagged mutagenesis was used. Two sets, each consisting of 378 signature-tagged mutants with a known transposon insertion site, were used in an experiment in planta. As a result, 67 mutants showing attenuated symbiotic phenotypes were identified, including most of the exo, fix, and nif mutants in the sets. For 38 mutants in genes previously not described to be involved in competitiveness or symbiosis in S. meliloti, attenuated competitiveness phenotypes were tested individually. A large part of these phenotypes was confirmed. Moreover, additional symbiotic defects were observed for mutants in several novel genes such as infection deficiency phenotypes (ilvI and ilvD2 mutants) or delayed nodulation (pyrE, metA, thiC, thiO, and thiD mutants).