An 11,955-bp region of the Borrelia burgdorferi chromosome containing all the genes encoding ribosomal RNA (rRNA) has been sequenced. The region contains a single gene encoding 16S rRNA and two genes encoding the 23S and 5S rRNAs. The sizes of the 16S, 23S and 5S rRNAs encoded by these genes are 1537, 2926 and 112 nucleotides, respectively. In addition, the genes encoding tRNA(Ala) and tRNA(Ile) are located in the intergenic spacer between the 16S and 23S rDNAs. The tDNAs do not encode the common CCA 3′ end which presumably must be added posttranscriptionally. All the genes are present in the same orientation, except for that encoding tRNA(Ile), which is transcribed from the opposite strand. The latter implies that the rDNAs are not transcribed as a single unit. The location of putative promoters and termination signals in the sequence suggest that the 16S rRNA and tRNA(Ala) are transcribed as a single unit, tRNA(Ile) is produced as an individual transcript and the 23S and 5S rDNAs are co-transcribed. Several of the features of this rDNA organization are unique, not having been described previously in any other eubacteria.