A Rhizobium leguminosarum biovar viciae strain lacking a 40 kb DNA region of the Sym plasmid pRL1IJ to the left (3' side) of gene nodE failed to nodulate Vicia sativa plants. Therefore this DNA region was investigated for the presence of additional nodulation genes. Complementation experiments indicated that the DNA region to the left (3' side) of nodE is functionally homologous between R. leguminosarum bv. viciae and R. leguminosarum bv. trifolii. In this DNA region, three nodulation genes were identified, nodT, nodM and nodL. TnphoA insertions in the nodT gene, about 4.5 kb to the left of nodE, lead to a delay in nodulation on Trifolium subterraneum, but not on V. sativa plants. TnphoA insertions in gene nodM have no detectable influence on nodulation. Finally, TnphoA insertions in the nodL gene affected nodulation so that only rarely nodules were induced on the inoculated plants. The nucleotide sequence of this gene is presented. On the basis of the sequence a membrane integrated protein is predicted with a molecular weight of 20.1 kDa. Microscopical analysis of the infection process by nodL mutants indicate a role for nodL in maintaining the stability of the infection thread. Experiments using transcriptional lacZ fusions suggest that nodL belongs to the same transcriptional unit as nodF,E. Very low expression of nodL seems to be sufficient for biological activity.