In all plant species studied to date, sucrose synthase occurs as multiple isoforms. The specific functions of the different isoforms are for the most part not clear. Six isoforms of sucrose synthase have been identified in the model legume Lotus japonicus, the same number as in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). The genes encoding these isoforms are differentially expressed in all plant organs examined, although one, LjSUS4, is only expressed in flowers. LjSUS1 is the most highly expressed in all plant organs tested, except root nodules, where LjSUS3 accounts for more than 60% of the total SUS transcripts. One gene, LjSUS2, produces two transcripts due to alternative splicing, a feature not observed in other species to date. We have isolated plants carrying ethyl methanesulfonate-induced mutations in several SUS genes by targeting-induced local lesions in genomes reverse genetics and examined the effect of null alleles of two genes, LjSUS1 and LjSUS3, on nodule function. No differences were observed between the mutants and wild-type plants under glasshouse conditions, but there was evidence for a nitrogen-starvation phenotype in the sus3-1 mutant and severe impairment of growth in the sus1-1/sus3-1 double mutant under specific environmental conditions. Nodules of sus3-1 mutant plants retained a capacity for nitrogen fixation under all conditions. Thus, nitrogen fixation can occur in L. japonicus nodules even in the absence of LjSUS3 (the major nodule-induced isoform of SUS), so LjSUS1 must also contribute to the maintenance of nitrogen assimilation.