Introduction of the subcloned nodO gene of Rhizobium sp. BR816 into R. meliloti 1021, R. leguminosarum bv. trifolii ANU843, and Azorhizobium caulinodans ORS571 significantly stimulated nodulation and infection of Leucaena leucocephala by these transconjugants. The BR816 nodO gene was also shown to suppress the nodulation defect of the nodU mutants of Rhizobium sp. NGR234 and R. tropici CIAT899 on L. leucocephala and of the nodE mutant of R. leguminosarum bv. trifolii ANU843 on white clover. Overproduction of NodO in BR816 extended its host range to Trifolium repens. While a Rhizobium sp. BR816 strain, mutated in nodO, had reduced nodulation and competitiveness on P. vulgaris, no altered phenotype was observed for this mutant on L. leucocephala. Analysis of the transcriptional activation of nodO in BR816 revealed enhanced expression in the presence of apigenin, under both aerobic and microaerobic conditions. Moreover, nodO was also expressed in the bacteroids of L. leucocephala nodules.