Rhizobia nodulation (nod) genes are involved in the synthesis of symbiotic signals, the Nod factors, which are mono-N-acylated chito-oligosaccharides. Nod factors elicit, in a specific manner, various plant responses on legume roots. In this report we address the question of the role of nodFEG genes in the synthesis of the acyl moiety of Rhizobium meliloti Nod factors. In a Nod factor-overproducing strain with the wild-type nod region, in addition to the delta 2,9-C16:2 and delta 2, 4,9-C16:3 acyl groups already described, delta 9-C16:1 was also found, together with a series of C18 to C26 (omega-1)-hydroxylated fatty acids. A deletion of nodE resulted in the absence of C16:2 and C16:3 fatty acids, which were replaced by vaccenic acid (delta 11-C18:1), but did not change the proportion of (omega-1)-hydroxylated fatty acids. A nodF deletion, non-polar with respect to nodE, resulted in the same alterations in the Nod factor N-acyl composition, showing that both nodF and nodE are required for the synthesis of the C16 polyunsaturated chains. In contrast, nodG mutations did not result in a detectable change in the Nod factor N-acyl moiety. When a plasmid carrying the nodFE genes of Rhizobium leguminosarum bv. viciae was introduced into R. meliloti nodFE- and nodFEG-deleted strains, Nod factors with polyunsaturated C18 fatty acids (C18:2, C18:3, and C18:4) could be detected. These results provide evidence that the molecular basis of allelic variation between the R. meliloti and R. leguminosarum bv. viciae host range nodFE genes lies in the fact that the two nodFE alleles specify the synthesis of unsaturated fatty acid substituents with a different carbon length.