A 135 kb long segment of the symbiotic region of the Rhizobium meliloti megaplasmid was mapped with the help of a Rhizobium meliloti gene library, made in the cosmid vehicle pJB8. A set of overlapping cosmid clones was used to identify the inserts in R-primes carrying megaplasmid sections, and to map 20 deletion mutations and 24 insertion mutations with Nod-or Fix-phenotypes. This led to the identification of DNA regions carrying nod or [ix (m/% genes. The results of this study correlate well with transcription data of nodule-specific expression of plasmid sequences. The nod mutations were localized in two groups. Using directed Tn5 mutagenesis, correlated physical-genetic maps for these regions were established. One nod gene cluster is about 2.5-3.0 kb in size and carries genes involved in root hair curling, a very early step in nodule formation. Mutations in these genes can be complemented by sym plasmids of other Rhizobium species, such as Rhi:ohium leguminosarum. We designate these genes as "'common" mud genes because mutations in them can be complemented by piasmids derived from different Rhi:ohhmt strains. The otl~er nod gene cluster consists of a 2 kb and a 1 kb long DNA segment, separated by a 1 kb region nonessential Ibr nodul:ation. These nod genes are probably involved in the host specificity of nodulation.