The 16S-23S intergenic spacers of Treponema pallidum subspecies pallidum, Nichols strain, and Treponema pallidum subspecies pertenue, Gauthier strain, have been cloned, characterized and sequenced. Isoleucine and alanine tRNA genes have been identified within the 16S-23S intergenic regions on separate alleles of 293 and 303 bases, respectively. The two alleles are present in both T.p pallidum and T.p. pertenue, and show no sequence differences between the bacterial subspecies. The ile-tRNA and ala-tRNA genes show 65% and 84% sequence identity, respectively, with the homologous genes of the related spirochete, Borrelia burgdorferi.