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DNA fragments containing the rRNA genes for Borrelia burgdorferi strain B31 were cloned in bacteriophage lambda EMBL3. A restriction map of the fragments was constructed and the organization of the rRNA genes was determined by Southern hybridization. One genomic DNA fragment contained a single copy of the rrs sequence and another cloned fragment contained both rrl and rrf sequences. The results revealed that the rrs gene is located separately from the set of rrl/rrf genes, suggesting that these rRNA genes are expressed independently in B. burgdorferi.