To determine the genetic diversity of cultivable and uncultivable spirochetes in the gingival crevice of a patient with severe periodontitis, partial 16S rRNA genes were cloned from PCR-amplified products of DNA and RNA extracted from a subgingival plaque sample. Approximately 500 bp were amplified in PCRs by using universally conserved primers with polylinker tails. Purified PCR products were cloned into Escherichia coli by using the plasmid vector pUC19. The resultant clone library was screened by colony hybridization with a radiolabeled, treponeme-specific oligonucleotide probe. The 16S rRNA inserts of 81 spirochetal clones were then sequenced by standard procedures. Sequences were compared with 16S rRNA sequences of 35 spirochetes, including the four known cultivable oral treponeme species. The analysis revealed an unexpected diversity of oral treponemes from a single patient. When 98% or greater sequence similarity was used as the definition of a species-level cluster, the clone sequences were found to represent 23 species. When 92% similarity was used as the definition, the clones fell into eight major groups, only two of which contained named species, Treponema vincentii and Treponema denticola, while Treponema pectinovorum and Treponema socranskii were not represented in any cluster. Seven of the 81 spirochetal clones were found to contain chimeric 16S rRNA sequences. In situ fluorescence hybridization with a fluorescein isothiocyanate-labeled oligonucleotide probe specific for one of the new species representing cluster 19 was used to identify cells of the target species directly in clinical samples.