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A molecular phylogenetic analysis was done of not-yet-cultured spirochaetes inhabiting the gut of the termite, Reticulitermes flavipes (Kollar). Ninety-eight clones of near-full-length spirochaetal 16S rDNA genes were classified by ARDRA pattern and by partial sequencing. All clones grouped within the genus Treponema, and at least 21 new species of Treponema were recognized within R. flavipes alone. Analysis of 190 additional clones from guts of Coptotermes formosanus Shiraki and Zootermopsis angusticollis (Hagen), as well as published data on clones from Cryptotermes domesticus (Haviland), Mastotermes darwiniensis Froggatt, Nasutitermes lujae (Wasmann) and Reticulitermes speratus (Kolbe), revealed a similar level of novel treponemal phylogenetic diversity in these representatives of five of the seven termite families. None of the clones was closely related (i.e. all bore ≤ 91% sequence similarity) to any previously recognized treponeme. The data also revealed the existence of two major phylogenetic groups of treponemes: one containing all of the currently known isolates of Treponema and a large number of phylotypes from the human gingival crevice, but only a minority of the termite gut spirochaete clones; another containing the majority of termite spirochaete clones and two Spirochaeta (S. caldaria and S. stenostrepta ), which, although free living, group within the genus Treponema on the basis of 16S rRNA sequence. Signature nucleotides that almost perfectly distinguished the latter group, herein referred to as the ‘termite cluster’, occurred at the following (E. coli numbering) positions: 289-G · C-311; A at 812; and an inserted nucleotide at 1273. The emerging picture is that the long-recognized and striking morphological diversity of termite gut spirochaetes is paralleled by their phylogenetic diversity and may reflect substantial physiological diversity as well.