Dr Robert Smibert from the Virginia Polytechnic Institute, USA, isolated and collected over 200 strains of oral treponemes over a 20-year period. Dr Smibert, Dr W. E. C. Moore and Dr L. V. Moore separated these isolates and reference strains into different groups on the basis of cellular fatty acid analysis. In this study, the 165 rRNA genes were sequenced for 47 strains that were representative of these groups. Five distinct species were identified on the basis of 165 rRNA sequence comparisons; two of these species are newly named and three have not yet been characterized. The first species, designated Treponema Smibert-1, was represented by the single strain D4B-1 and was later identified as the newly described Treponema maltophilum. However, strain D4B-1 possessed a different flagellar arrangement to that of T.maltophilum . The second species, Treponema S m i bert-2, was represented by nine isolates that possessed identical 165 rRNA gene sequences. The closest relatives of this species were Treponema Smibert-3 and Treponema Smibert-4 a t approximately 90 YO sequence similarity. Within Treponema Smibert-2, there was no correlation between phylogenetic analysis and cellular fatty acid analysis since six different cellular fatty acid groups represented the nine strains. Treponema Smibert-3 (strain D36ER-1) and Treponema Smibert-4 (D62CR-12) were each represented by only a single strain and were closely related to each other at 98% sequence similarity. Strain D36ER-1 of Treponema Smibert-3 was identified as belonging to the not-yet-cultivated phylotype 20 [Choi, B. K. , Paster, B. J. , Dewhirst, F. E. & Gebel, U. 8. (1994). Infect Immun 62,1889-18951. Strain D62CR-I2 of Treponema Smibert-4 was nearly identical in sequence to the newly described Treponema amylovorum. The fifth species, Treponema Smibert-5, was represented by a single strain, D120CR-1, and was closely related at about 98% sequence similarity to the three subspecies of Treponema socranskii. The phylogenetic analyses of strains of Treponema vincentii and of subspecies of T. socranskii are also reported. The closest oral relatives of T. vincentii were Treponema medium a t 987 O/ O sequence similarity and Treponema denticola a t 91.5 YO sequence similarity. T. socranskii subspp.socranskii, buccale and paredis formed three separate phylogenetic branches with sequence similarities of about 98% to each other. The closest relative of the subspecies of T. socranskii and of Smibert-5 was Smibert-2 a t about 86% sequence similarity. Historic reference strains Fuji, ' Treponema ambigua I , Fm, Ichelson-2, N-39, TD2, TRRD, MRB, IPP, Jethro and T32A, as well as an unknown strain designated only as Treponema oralis, were identified as strains of T. denticola. Reference strains Fuji, Jethro, T32A and IPP plus three isolates of the Smibert collection were also contaminated with a mycoplasma as determined by 165 rRNA comparative analysis. Consequently, spirochaetal cultures should be screened for mycoplasmas. There are presently a t least ten species of cultivable o...