Most nodulatlon genes of the broad host-range Rhizobium sp. NGR234 are located on the 536 kb symbiotic plasmid. Complete sequencing of the replicon revealed over 400 open-reading frames (ORF), many of which correspond to known genes. Combined physical/genetic analysis of the host specificity of nodulation (hsn) locus lll revealed many nodulation genes, some of which are essential for Nod-factor production (nodABQ and others which modify the Nod-factor structure in specific ways (noeE). When mutated, two other genes, ORF3 and fixF gave Fix⁻ phenotypes on Vigna unguiculata. fixF has a nuclear localisation sequence (NLS) which is functional in nuclear targeting in tobacco protoplasts. Mutations in fixF abolish production of a novel, rhamnose-rich lipopolysaccharide that is produced under conditions of flavonoid induction.