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Genomic DNAs of 14 strains from seven species of the spirochete Leptospira were resistant to cleavage by the restriction endonuclease RsaI 1 (5'-GTAC). A modified base comigrating with m4C was detected by chromatography. Genomic DNAs from other spirochetes, Borrelia group VS461, and Serpulina strains were not resistant to h a 1 digestion. Modification at 5'-GTAm4C may occur in most or all strains of all species of Leptospira but not in all genera of spirochetes. Genus-wide DNA modification has rarely been observed in bacteria.