In order to study the function of nodulin genes and the activity of their promoters, the Agrobacterium rhizogenes transformation strategy was adopted for Sesbania rostrata. Two protocols were selected to generate S. rostrata transgenic roots that can be nodulated efficiently after application of Azorhizobium caulinodans, ORS571. Cotransformation frequencies of 22 and 72% were obtained with the first and second protocol, respectively. The transgenic root nodules showed no apparent differences when compared with the wild-type root nodules. The screening procedure for cotransformation of binary T-DNA in transgenic roots and root nodules was optimized by using an enhanced green-fluorescent protein construct. The A. rhizogenes transformation system was used to analyze the 35S promoter-driven expression of reporter genes in developing root nodules.