In Rhizobium leguminosarum both the C,-dicarboxylate transport system and wild-type lipopolysaccharide layer (LPS) are essential for nitrogen fixation. A TnS mutant (RU301) of R. leguminosarum bv. viciae 3841, was isolated that is only able to synthesize LPS II, which lacks the 0-antigen. Strain RU301 exhibits a rough colony morphology, flocculates in culture and is unable to swarm in TY agar. It also fails to grow on organic acids, sugars or TY unless the concentration of calcium or magnesium is elevated above that normally required for growth. The defects in the LPS and growth in strain RU301 were complemented by a series of cosmids from a strain 3841 cosmid library (pRU3020-pRU3022) and a cosmid from R. leguminosarum bv. phaseoli 8002 (plJ1848). The transposon insertion in strain RU301 was shown to be located in a 3 kb EcoRl fragment by Southern blotting and cloning from the chromosome. Sub-cloning of plJ1848 demonstrated that the gene disrupted by the transposon in strain RU301 is located on a 2-4 kb EcoRI-Pstl fragment (pRU74). R. leguminosarum bv. viciae VF39-C86, which is one of four LPS mutants previously isolated by U. B. Priefer (1989, J Bacteriol 171, 6161-6168), was also complemented by sub-clones of plJ1848 but not by pRU74, suggesting the mutation is in a gene adjacent to that disrupted in strain RU301. Complementation and Southern analysis indicate that the region contained in plJ1848 does not correspond to any other cloned Ips genes. Two dctA mutants, RU436 and RU437, were also complemented by plJ1848 and pRU3020. Mapping of plJ1848 and Southern blotting of plasmid-deleted strains of R. leguminosanrm revealed that dctD and the region mutated in strain RU301 are located approximately 10 kb apart on the chromosome. Analysis of homogenotes demonstrated that there is not a large region important in calcium utilization, organic acid metabolism or LPS biosynthesis located between the gene disrupted in strain RU301 and dctD. Strain VF39C-86 also required an elevated concentration of calcium for growth on succinate, while strains mutated in the a-chromosomal or /?-plasmid group of Ips genes grew at the same calcium concentrations as the wild type, demonstrating that the additional calcium requirement is not a property of all LPS rough mutants. Strain RU301 nodulates peas, but does not reduce acetylene, demonstrating that the gene mutated in this strain is essential for nitrogen fixation.