Screening of derivatives of Rhizobium etli KIM5s randomly mutagenized with mTn5SSgusA30 resulted in the identification of strain KIM-G1. Its rough colony appearance, flocculation in liquid culture, and Ndv Fix phenotype were indicative of a lipopolysaccharide (LPS) defect. Electrophoretic analysis of cell-associated polysaccharides showed that KIM-G1 produces only rough LPS. Composition analysis of purified LPS oligosaccharides from KIM-G1 indicated that it produces an intact LPS core trisaccharide (-D-GalA-14[-D-GalA-15]-Kdo) and tetrasaccharide (-D-Gal-16[-D-GalA-14]--D-Man-15Kdo), strongly suggesting that the transposon insertion disrupted a locus involved in O-antigen biosynthesis. Five monosaccharides (Glc, Man, GalA, 3-O-Me-6-deoxytalose, and Kdo) were identified as the components of the repeating O unit of the smooth parent strain, KIM5s. Strain KIM-G1 was complemented with a 7.2-kb DNA fragment from KIM5s that, when provided in trans on a broad-host-range vector, restored the smooth LPS and the full capacity of nodulation and fixation on its host Phaseolus vulgaris. The mTn5 insertion in KIM-G1 was located at the N terminus of a putative -glycosyltransferase, which most likely had a polar effect on a putative -glycosyltransferase located downstream. A third open reading frame with strong homology to sugar epimerases and dehydratases was located upstream of the insertion site. The two glycosyltransferases are strain specific, as suggested by Southern hybridization analysis, and are involved in the synthesis of the variable portion of the LPS, i.e., the O antigen. This newly identified LPS locus was mapped to a 680-kb plasmid and is linked to the lps2 gene recently reported for R. etli CFN42.