A physical map of the genome of Rhizobium meliloti 1021 is presented. The physical sizes of the three replicons in this genome had previously been determined and are as follows: the chromosome, 3.4 Mb; pSym-b, 1.7 Mb; and pSym-a, 1.4 Mb. The physical maps for this GC-rich genome contain AT-rich restriction sites for SwaI (5'-TAAATTTA-3'), PacI (5'-TTAATTAA-3'), PmeI (5'-GTTTAAAC-3'), and, for pSym-b, SpeI (5'-ACTAGT-3'). In addition, the endonuclease I-CeuI cleaved the 23S rRNA genes in this genome, and perhaps in most eubacterial genomes. I-CeuI digestion and polymerase chain reaction amplification of rrn regions were used to determine that there are at least three rrn loci in R. meliloti, all of which are located on the chromosome. The orientation of the rrn loci was determined by Southern blotting with probes from rrn sequences located 5' and 3' to the I-CeuI site. The rrn loci are clustered in one part of the chromosome and are oriented so that transcription will occur away from a single point in the circle, as observed for the origin of replication in the Escherichia coli and Salmonella typhimurium chromosomes. Fifteen genes that had been tagged by Tn5 insertion were localized to fragments on the chromosome physical map by using the IS50 as a probe in Southern blots. In addition, glt and gap were placed on the physical map by using Southern hybridization with cloned genes. The fortuitous occurrence of SpecI site in Tn5-233 was used to physically map 10 genetically mapped Tn5-233 integrations on pSym-b and to anchor the physical map to the genetic map. Finally, we demonstrate the usefulness of the map by localizing a total of 12 previously unmapped transposon insertions in the genome. This is the first physical map of the genome of a multireplicon member of the family Rhizobiaceae as well as the first physical map of a Rhizobium chromosome.